Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair.

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

The role of intraamniotic inflammation in threatened midtrimester miscarriage.

BACKGROUND: The assessment and management of patients with threatened midtrimester miscarriage is a clinical challenge because the etiology of this condition is poorly understood. OBJECTIVE: This study aimed to examine the frequency of intraamniotic infection or inflammation and the effect of antibiotics in patients presenting with regular uterine contractions and intact membranes before 20 weeks of gestation. STUDY DESIGN: This retrospective study comprised patients who met the following criteria: (1) singleton gestation, (2) gestational age before 20 weeks, (3) the presence of regular uterine contractions confirmed by a tocodynamometer (8 or more contractions in 60 minutes), (4) intact amniotic membranes, and (5) transabdominal amniocentesis performed for the evaluation of the microbiologic and inflammatory status of the amniotic cavity. Samples of amniotic fluid were cultured for aerobic and anaerobic bacteria and genital mycoplasmas, and polymerase chain reaction was performed to detect Ureaplasma species. Amniotic fluid was tested for white blood cell counts and matrix metalloproteinase-8 concentrations to diagnose intraamniotic inflammation. Patients with intraamniotic inflammation, or intraamniotic infection, were treated with antibiotics (a combination of ceftriaxone, clarithromycin, and metronidazole). Treatment success was defined as the resolution of intraamniotic infection/inflammation at the follow-up amniocentesis or delivery after 34 weeks of gestation. RESULTS: 1) Intraamniotic inflammation was present in 88% (15/17) of patients, whereas infection was detectable in only 2 cases; 2) objective evidence of resolution of intraamniotic inflammation after antibiotic treatment was demonstrated in 100% (4/4) of patients who underwent a follow-up amniocentesis; 3) 30% (5/15) of women receiving antibiotics delivered after 34 weeks of gestation (3 of the 5 patients had a negative follow-up amniocentesis, and 2 of the women were without a follow-up amniocentesis); 4) the overall treatment success of antibiotics was 40% (6/15; 4 cases of objective evidence of resolution of intra-amniotic inflammation and 5 cases of delivery after 34 weeks of gestation). CONCLUSION: The prevalence of intraamniotic inflammation in patients who presented with a threatened midtrimester miscarriage was 88% (15/17), and, in most cases, microorganisms could not be detected. Antibiotic treatment, administered to patients with intraamniotic inflammation, was associated with either objective resolution of intraamniotic inflammation or delivery after 34 weeks of gestation in 40% (6/15) of the cases.

Landscape of Cellular Bioeffects Triggered by Ultrasound-Induced Sonoporation.

Sonoporation is the process of transient pore formation in the cell membrane triggered by ultrasound (US). Numerous studies have provided us with firm evidence that sonoporation may assist cancer treatment through effective drug and gene delivery. However, there is a massive gap in the body of literature on the issue of understanding the complexity of biophysical and biochemical sonoporation-induced cellular effects. This study provides a detailed explanation of the US-triggered bioeffects, in particular, cell compartments and the internal environment of the cell, as well as the further consequences on cell reproduction and growth. Moreover, a detailed biophysical insight into US-provoked pore formation is presented. This study is expected to review the knowledge of cellular effects initiated by US-induced sonoporation and summarize the attempts at clinical implementation.

Actin Cytoskeletal Dynamics in Single-Cell Wound Repair.

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.

Risk factors and maternal outcomes following preterm premature rupture of membrane in the second trimester of gestation.

PURPOSE: To characterize the population of women who underwent mid-trimester preterm premature rupture of membrane (PPROM) in a country where mid-trimester abortions are legal and available. METHODS: A retrospective cross-sectional cohort study was conducted at a tertiary referral hospital, during 2013-2016. Mid-trimester defined as gestational age 13 + 0 to 23 + 6 weeks. Rupture of membrane was defined by documentation of fluid passing through the cervix on sterile speculum examination, and a positive Nitrazine (Bristol-Myers Squibb, Princeton, NJ) or erning test. All records were evaluated for medical history, laboratory data, postnatal examination, and autopsy findings, and a database was constructed. RESULTS: A total of 61 women were hospitalized for mid-trimester PPROM during the study period. Mean maternal age was 32 ± 5.98, range 20-45 years old. The majority (50, 82%) of patients decided to terminate their pregnancy before reaching the limit of viability at 24 weeks gestation. The overall prognosis of pregnancies reaching term was better than expected, with six (9.8%) patients delivering live babies and four of them born at term (36 ± 5 to 40 ± 6 weeks gestation), all after PPROM following amniocentesis or selective fetal reduction. A total of 60% of women with hypothyroidism had unbalanced TSH levels above 4.0 mIU/L prior to their pregnancy. A notable number of women (15, 24.6%) had PPROM following a pregnancy achieved by assisted reproductive technology (ART). CONCLUSIONS: Most women with diagnosed mid-trimester PPROM opted for pregnancy termination before the limit of viability when granted the choice. Possible risk factors for early PPROM are unbalanced hypothyroidism and ART. PPROM following amniocentesis can in some cases reseal and reach term, suggesting conservative treatment is a reasonable management for those cases.

Autocrine purinergic signaling stimulated by cell membrane disruption is involved in both cell membrane repair and adaptive response in MDCK cells.

Disruption and repair of plasma membranes is normally observed in many animal tissues. Recent studies demonstrated that wounding of Madin-Darby canine kidney cells potentiates membrane repair in cells adjacent to wounded cells via paracrine purinergic signaling. The present study demonstrated that cyclic adenosine monophosphate signaling in a wounded cell was induced by autocrine purinergic signaling, and protein kinase A potentiates membrane resealing for repeated wounds in those cells. Furthermore, the present study revealed that an increase in the intracellular free Ca2+ concentration upon cell membrane disruption was not only due to Ca2+ influx through the wound site, but also because of autocrine purinergic signaling. Although the influx of extracellular Ca2+ is essential for membrane resealing, the present study suggested that an increase in the intracellular free Ca2+ concentration induced by autocrine signaling accelerates membrane resealing of the initial cell membrane disruption.

In vitro cellular viability studies on a concentrated surfactant-based wound dressing.

In this study, three cellular cytotoxic assays (direct contact assay, extraction assay, and cell insert assay) were applied to evaluate the effects of a concentrated surfactant gel preserved with antimicrobials and a concentrated surfactant gel with 1% silver sulfadiazine on both the mouse fibroblast cell line L929 and human dermal fibroblasts (HDFa). Also, the in vitro wound model was wounded by a 100 µL pipette tip and used to assess cell migration and wound closure after treatment with both gels. A needle-scratched membrane disruption model was used to preliminarily evaluate membrane stabilisation and the membrane-resealing effects of concentrated surfactant gels. It was demonstrated that the concentrated surfactant gel preserved with antimicrobials was not toxic to both L929 and HDFa. However, the concentrated surfactant gel with 1% silver sulfadiazine demonstrated a degree of cytotoxicity to both cell types. After treatment with a concentrated surfactant gel preserved with antimicrobials, cell movement to close the scratch gap was enhanced at 24 and 48 hours. The results also showed that cells treated with the concentrated surfactant gel preserved with antimicrobials decreased cell necrosis and improved cell resistance of the f-actin rearrangement after a needle scratch. The results demonstrated that a concentrated surfactant gel preserved with antimicrobials is non-cytotoxic and has ability to accelerate wound closure by enhancing cell mobility. Furthermore, the concentrated surfactant gel appeared to stabilise the plasma membrane and demonstrated a resealing ability and helped to retain the plasma membrane integrity and enhanced wound healing.

ESCRT: nipping the wound in the bud?

Rapid repair of plasma membrane wounds is critical for cellular survival. Exocytic patches, membrane tension reduction and endocytosis were previously proposed to mediate resealing. A recent study implicating the ESCRT complex adds to the growing evidence that repair involves removal of damaged plasma membrane, and not simply patching the wound.

Above the fray: Surface remodeling by secreted lysosomal enzymes leads to endocytosis-mediated plasma membrane repair.

The study of plasma membrane repair is coming of age. Mirroring human adolescence, the field shows at the same time signs of maturity and significant uncertainty, confusion and skepticism. Here we discuss concepts that emerged from experimental data over the years, some of which are solidly established while others are still subject to different interpretations. The firmly established concepts include the critical requirement for Ca(2+) in wound repair, and the role of rapid exocytosis of intracellular vesicles. Lysosomes are being increasingly recognized as the major vesicles involved in injury-induced exocytosis in many cell types, as a growing number of laboratories detect markers for these organelles on the cell surface and lysosomal hydrolases in the supernatant of wounded cells. The more recent observation of massive endocytosis following Ca(2+)-triggered exocytosis initially came as a surprise, but this finding is also being increasingly reported by different groups, shifting the discussion to the mechanisms by which endocytosis promotes repair, and whether it operates or not in parallel with the shedding of membrane blebs. We discuss how the abundant intracellular vesicles that undergo homotypic fusion close to wound sites, previously interpreted as exocytic membrane patches, actually acquire extracellular tracers demonstrating their endocytic origin. We also suggest that an initial, temporary patch that prevents cytosol loss until the bilayer is restored might result not from vesicular fusion, but from rapid Ca(2+)-dependent crosslinking and aggregation of cytosolic proteins. Finally, we propose that cell surface remodeling, orchestrated by the extracellular release of lysosomal hydrolases and perhaps also cytosolic molecules, may represent a key aspect of the plasma membrane repair mechanism that has received little attention so far.

The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds.

The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca(2+)-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca(2+)-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery.

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

Annexin-A5 promotes membrane resealing in human trophoblasts.

Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a µm²-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Membrane Tension Regulation is Required for Wound Repair.

Disruptions of the eukaryotic plasma membrane due to chemical and mechanical challenges are frequent and detrimental and thus need to be repaired to maintain proper cell function and avoid cell death. However, the cellular mechanisms involved in wound resealing and restoration of homeostasis are diverse and contended. Here, it is shown that clathrin-mediated endocytosis is induced at later stages of plasma membrane wound repair following the actual resealing of the wound. This compensatory endocytosis occurs near the wound, predominantly at sites of previous early endosome exocytosis which is required in the initial stage of membrane resealing, suggesting a spatio-temporal co-ordination of exo- and endocytosis during wound repair. Using cytoskeletal alterations and modulations of membrane tension and membrane area, membrane tension is identified as a major regulator of the wounding-associated exo- and endocytic events that mediate efficient wound repair. Thus, membrane tension changes are a universal trigger for plasma membrane wound repair modulating the exocytosis of early endosomes required for resealing and subsequent clathrin-mediated endocytosis acting at later stages to restore cell homeostasis and function.

The Dysferlinopathies Conundrum: Clinical Spectra, Disease Mechanism and Genetic Approaches for Treatments.

Dysferlinopathies refer to a spectrum of muscular dystrophies that cause progressive muscle weakness and degeneration. They are caused by mutations in the DYSF gene, which encodes the dysferlin protein that is crucial for repairing muscle membranes. This review delves into the clinical spectra of dysferlinopathies, their molecular mechanisms, and the spectrum of emerging therapeutic strategies. We examine the phenotypic heterogeneity of dysferlinopathies, highlighting the incomplete understanding of genotype-phenotype correlations and discussing the implications of various DYSF mutations. In addition, we explore the potential of symptomatic, pharmacological, molecular, and genetic therapies in mitigating the disease's progression. We also consider the roles of diet and metabolism in managing dysferlinopathies, as well as the impact of clinical trials on treatment paradigms. Furthermore, we examine the utility of animal models in elucidating disease mechanisms. By culminating the complexities inherent in dysferlinopathies, this write up emphasizes the need for multidisciplinary approaches, precision medicine, and extensive collaboration in research and clinical trial design to advance our understanding and treatment of these challenging disorders.

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage.

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+ -evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

Monitoring optoporated process on mammalian cells by real-time measurement of membrane resealing time.

Significance: Resealing time based loading efficiency of optoporation is the key parameter for drug or gene delivery. This work describes a comparatively simple optical approach to directly measure the cell membrane resealing time of the gold nanoparticle mediated photoporation. Aim: To establish a membrane potential detection optical system, which can provide a direct measurement of resealing time of the optoporated cells. Approach: Voltage sensitive dye has been used to label the gold nanoparticle covered cell before laser activation and the resealing time was estimated from the voltage change due to the fluorescence light intensity change before and after laser activation. The approach has been validated by the simulated data based on diffusion model and Monte Carlo simulation and the experimental data obtained from a flow cytometry analysis. Results: The measured resealing time after perforation varied from 28.6 to 163.8 s on Hela cells when the irradiation fluence was increased, with a correlation coefficient (R2) of 0.9938. This result is in agreement with the resealing time (1-2 min) of photothermal porated Hela cells measured by electrical impedance method. The intracellular delivery efficiency of extracellular macromolecular under the same irradiation fluence depends mainly on diffusion velocity rather than pore size. Conclusion: The method described here can be used to directly measure resealing time of optoporated cells for accurately estimating the loading efficiency on discovering the mechanism of optoporation.

Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles.

To develop new strategies for improving the efficacy and biosafety of sonoporation-based macromolecule delivery, it is essential to understand the mechanisms underlying plasma membrane re-sealing and function recovery of the cells perforated by ultrasound-driven microbubbles. However, we lack a clear understanding of the spatiotemporal dynamics of the disrupted actin cytoskeleton and its role in the re-sealing of sonoporated cells. Here we used a customized experimental setup for single-pulse ultrasound (133.33-µs duration and 0.70-MPa peak negative pressure) exposure to microbubbles and for real-time recording of single-cell (human umbilical vein endothelial cell) responses by laser confocal microscopic imaging. We found that in reversibly sonoporated cells, the locally disrupted actin cytoskeleton, which was spatially correlated with the perforated plasma membrane, underwent three successive phases (expansion; contraction and re-sealing; and recovery) to re-model and that each phase of the disrupted actin cytoskeleton was approximately synchronized with that of the perforated plasma membrane. Moreover, compared with the closing time of the perforated plasma membrane, the same time was used for the re-sealing of the actin cytoskeleton in mildly sonoporated cells and a longer time was required in moderately sonoporated cells. Further, the generation, directional migration, accumulation and re-polymerization of globular actin polymers during the three phases drove the re-modeling of the actin cytoskeleton. However, in irreversibly sonoporated cells, the actin cytoskeleton, which underwent expansion and no contraction, was progressively de-polymerized and could not be re-sealed. Finally, we found that intracellular calcium transients were essential for the recruitment of globular actin and the re-modeling of the actin cytoskeleton. These results provide new insight into the role of actin cytoskeleton dynamics in the re-sealing of sonoporated cells and serve to guide the design of new strategies for sonoporation-based delivery.

Faster calcium recovery and membrane resealing in repeated sonoporation for delivery improvement.

In sonoporation-based macromolecular delivery, repetitive microbubble cavitation in the bloodstream results in repeated sonoporation of cells or sonoporation of non-sonoporated neighboring cells (i.e., adjacent to the sonoporated host cells). The resealing and recovery capabilities of these two types of sonoporated cells affect the efficiency and biosafety of sonoporation-based delivery. Therefore, an improved understanding of the preservation of viability in these sonoporated cells is necessary. Using a customized platform for single-pulse ultrasound exposure (pulse length 13.33 µs, peak negative pressure 0.40 MPa, frequency 1.5 MHz) and real-time recording of membrane perforation and intracellular calcium fluctuations (using propidium iodide and Fluo-4 fluorescent probes, respectively), spatiotemporally controlled sonoporation was performed to administer first and second single-site sonoporations of a single cell or single-site sonoporation of a neighboring cell. Two distinct intracellular calcium changes, reversible and irreversible calcium fluctuations, were identified in cells undergoing repeat reversible sonoporation and in neighboring cells undergoing reversible sonoporation. In addition to an increased proportion of reversible calcium fluctuations that occurred with repeated sonoporation compared with that in the initial sonoporation, repeated sonoporation resulted in significantly shorter calcium fluctuation durations and faster membrane resealing than that produced by initial sonoporation. Similarly, compared with those in sonoporated host cells, the intracellular calcium fluctuation recovery and membrane perforation resealing times were significantly shorter in sonoporated neighboring cells. These results demonstrated that the function recovery and membrane resealing capabilities after a second sonoporation or sonoporation of neighboring cells were potentiated in the short term. This could aid in sustaining the long-term viability of sonoporated cells, therefore improving delivery efficiency and biosafety. This investigation provides new insight into the resealing and recovery capabilities in re-sonoporation of sonoporated cells and sonoporation of neighboring cells and can help develop safe and efficient strategies for sonoporation-based drug delivery.

Tissue Barrier-on-Chip: A Technology for Reproducible Practice in Drug Testing.

One key application of organ-on-chip systems is the examination of drug transport and absorption through native cell barriers such the blood-brain barrier. To overcome previous hurdles related to the transferability of existing static cell cultivation protocols and polydimethylsiloxane (PDMS) as the construction material, a chip platform with key innovations for practical use in drug-permeation testing is presented. First, the design allows for the transfer of barrier-forming tissue into the microfluidic system after cells have been seeded on porous polymer or Si3N4 membranes. From this, we can follow highly reproducible models and cultivation protocols established for static drug testing, from coating the membrane to seeding the cells and cell analysis. Second, the perfusion system is a microscopable glass chip with two fluid compartments with transparent embedded electrodes separated by the membrane. The reversible closure in a clamping adapter requires only a very thin PDMS sealing with negligible liquid contact, thereby eliminating well-known disadvantages of PDMS, such as its limited usability in the quantitative measurements of hydrophobic drug molecule concentrations. Equipped with tissue transfer capabilities, perfusion chamber inertness and air bubble trapping, and supplemented with automated fluid control, the presented system is a promising platform for studying established in vitro models of tissue barriers under reproducible microfluidic perfusion conditions.

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis.

"Trim-Away" technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.