Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease.

The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation.

CD11b(+) dendritic cells (DCs) seem to be specialized for presenting antigens via major histocompatibility (MHC) class II complexes to stimulate helper T cells, but the genetic and regulatory basis for this is not established. Conditional deletion of Irf4 resulted in loss of CD11b(+) DCs, impaired formation of peptide-MHC class II complexes and defective priming of helper T cells but not of cytotoxic T lymphocyte (CTL) responses. Gene expression and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analyses delineated an IRF4-dependent regulatory module that programs enhanced MHC class II antigen presentation. Expression of the transcription factor IRF4 but not of IRF8 restored the ability of IRF4-deficient DCs to efficiently process and present antigen to MHC class II-restricted T cells and promote helper T cell responses. We propose that the evolutionary divergence of IRF4 and IRF8 facilitated the specialization of DC subsets for distinct modes of antigen presentation and priming of helper T cell versus CTL responses.

Specification of type 2 innate lymphocytes by the transcriptional determinant Gfi1.

Type 2 innate lymphoid cells (ILC2 cells) participate in host defense against helminth parasites and in allergic inflammation. Given their functional relatedness to type 2 helper T cells (T(H)2 cells), we explored whether Gfi1 acts as a shared transcriptional determinant in ILC2 cells. Gfi1 promoted the development of ILC2 cells and controlled their responsiveness during infection with Nippostrongylus brasiliensis and protease allergen-induced lung inflammation. Gfi1 'preferentially' regulated the responsiveness of ILC2 cells to interleukin 33 (IL-33) by directly activating Il1rl1, which encodes the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in impaired expression of the transcription factor GATA-3 and a dysregulated genome-wide effector state characterized by coexpression of IL-13 and IL-17. Our findings establish Gfi1 as a shared determinant that reciprocally regulates the type 2 and IL-17 effector states in cells of the innate and adaptive immune systems.

Bleomycin induces senescence and repression of DNA repair via downregulation of Rad51.

BACKGROUND: Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. METHODS: Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. RESULTS: Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin-induced mouse pulmonary fibrosis model. CONCLUSIONS: Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway.

Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE(+)) cells is poorly understood. A model of the differentiation of IgE(+) B cells proposes that IgE(+) cells develop through a germinal-center IgG1(+) intermediate and that IgE memory resides in the compartment of IgG1(+) memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE(+) cells developed through a germinal-center IgE(+) intermediate to form IgE(+) memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.

Downregulation of SMAD4 protects HaCaT cells against UVB-induced damage and oxidative stress through the activation of EMT.

As a member of the SMAD family, SMAD4 plays a crucial role in several cellular biological processes. However, its function in UVB radiation-induced keratinocyte damage is not yet clarified. Our study aims to provide mechanistic insight for the development of future UVB protective therapies and therapeutics involving SMAD4. HaCaT cells were treated with UVB, and the dose dependence and time dependence of UVB were measured. The cell function of UVB-treated HaCaT cells and the activity of epithelial-mesenchymal transition (EMT) after overexpression or silencing of SMAD4 was observed by flow cytometry, quantitative reverse transcription PCR (qRT-PCR) and Western Blots (WB). We found that a significant decrease in SMAD4 was observed in HaCaT cells induced by UVB. Our data confirm SMAD4 as a direct downstream target of miR-664. The down-regulation of SMAD4 preserved the viability of the UVB-treated HaCaT cells by inhibiting autophagy or apoptosis. Furthermore, the silencing of SMAD4 activated the EMT process in UVB-treated HaCaT cells. Down-regulation of SMAD4 plays a protective role in UVB-treated HaCaT cells via the activation of EMT.

The COP9 signalosome stabilized MALT1 promotes Non-Small Cell Lung Cancer progression through activation of NF-κB pathway.

MALT1 has been implicated as an upstream regulator of NF-κB signaling in immune cells and tumors. This study determined the regulatory mechanisms and biological functions of MALT1 in non-small cell lung cancer (NSCLC). In cell culture and orthotopic xenograft models, MALT1 suppression via gene expression interference or protein activity inhibition significantly impaired malignant phenotypes and enhanced radiation sensitivity of NSCLC cells. CSN5, the core subunit of COP9 signalosome, was firstly verified to stabilize MALT1 via disturbing the interaction with E3 ligase FBXO3. Loss of FBXO3 in NSCLC cells reduced MALT1 ubiquitination and promoted its accumulation, which was reversed by CSN5 interference. An association between CSN5/FBXO3/MALT1 regulatory axis and poor prognosis in NSCLC patients was identified. Our findings revealed the detail mechanism of continuous MALT1 activation in NF-κB signaling, highlighting its significance as predictor and potential therapeutic target in NSCLC.

Error detection for radiotherapy planning validation based on deep learning networks.

BACKGROUND: Quality assurance (QA) of patient-specific treatment plans for intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) necessitates prior validation. However, the standard methodology exhibits deficiencies and lacks sensitivity in the analysis of positional dose distribution data, leading to difficulties in accurately identifying reasons for plan verification failure. This issue complicates and impedes the efficiency of QA tasks. PURPOSE: The primary aim of this research is to utilize deep learning algorithms for the extraction of 3D dose distribution maps and the creation of a predictive model for error classification across multiple machine models, treatment methodologies, and tumor locations. METHOD: We devised five categories of validation plans (normal, gantry error, collimator error, couch error, and dose error), conforming to tolerance limits of different accuracy levels and employing 3D dose distribution data from a sample of 94 tumor patients. A CNN model was then constructed to predict the diverse error types, with predictions compared against the gamma pass rate (GPR) standard employing distinct thresholds (3%, 3 mm; 3%, 2 mm; 2%, 2 mm) to evaluate the model's performance. Furthermore, we appraised the model's robustness by assessing its functionality across diverse accelerators. RESULTS: The accuracy, precision, recall, and F1 scores of CNN model performance were 0.907, 0.925, 0.907, and 0.908, respectively. Meanwhile, the performance on another device is 0.900, 0.918, 0.900, and 0.898. In addition, compared to the GPR method, the CNN model achieved better results in predicting different types of errors. CONCLUSION: When juxtaposed with the GPR methodology, the CNN model exhibits superior predictive capability for classification in the validation of the radiation therapy plan on different devices. By using this model, the plan validation failures can be detected more rapidly and efficiently, minimizing the time required for QA tasks and serving as a valuable adjunct to overcome the constraints of the GPR method.

Anatomical changes and dosimetric analysis of the neck region based on FBCT for nasopharyngeal carcinoma patients during radiotherapy.

BACKGROUND: The study aimed to investigate anatomical changes in the neck region and evaluate their impact on dose distribution in patients with nasopharyngeal carcinoma (NPC) undergoing intensity modulated radiation therapy (IMRT). Additionally, the study sought to determine the optimal time for replanning during the course of treatment. METHODS: Twenty patients diagnosed with NPC underwent IMRT, with weekly pretreatment kV fan beam computed tomography (FBCT) scans in the treatment room. Metastasized lymph nodes in the neck region and organs at risk (OARs) were redelineation using the images from the FBCT scans. Subsequently, the original treatment plan (PLAN0) was replicated to each FBCT scan to generate new plans labeled as PLAN 1-6. The dose-volume histograms (DVH) of the new plans and the original plan were compared. One-way repeated measure ANOVA was utilized to establish threshold(s) at various time points. The presence of such threshold(s) would signify significant change(s), suggesting the need for replanning. RESULTS: Progressive volume reductions were observed over time in the neck region, the gross target volume for metastatic lymph nodes (GTVnd), as well as the submandibular glands and parotids. Compared to PLAN0, the mean dose (Dmean) of GTVnd-L significantly increased in PLAN5, while the minimum dose covering 95% of the volume (D95%) of PGTVnd-L showed a significant decrease from PLAN3 to PLAN6. Similarly, the Dmean of GTVnd-R significantly increased from PLAN4 to PLAN6, whereas the D95% of PGTVnd-R exhibited a significant decrease during the same period. Furthermore, the dose of bilateral parotid glands, bilateral submandibular glands, brainstem and spinal cord was gradually increased in the middle and late period of treatment. CONCLUSION: Significant anatomical and dosimetric changes were noted in both the target volumes and OARs. Considering the thresholds identified, it is imperative to undertake replanning at approximately 20 fractions. This measure ensures the delivery of adequate doses to target volumes while mitigating the risk of overdosing on OARs.

IL-17C regulates the innate immune function of epithelial cells in an autocrine manner.

Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the T(H)17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate-induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

Stroke as initial manifestation of non-small cell lung cancer with Trousseau syndrome.

OBJECTIVE: Stroke is a rare but fatal complication of advanced cancer with Trousseau syndrome, especially as initial symptoms. Here, we report the clinical characteristics, treatment, and prognosis of patients with non-small cell lung cancer (NSCLC) who initially presenting with acute multiple cerebral infarction. METHODS: The clinical characteristics, imaging, treatment, and oncological outcomes of 10 patients diagnosed with Trousseau syndrome and NSCLC between 2015 and 2021 at Guangdong Sanjiu Brain Hospital were retrospectively collected and analyzed. The clinical course of two typical cases were presented. RESULTS: All 10 patients with pathologically confirmed lung adenocarcinoma initially presented with neurological symptoms, including hemiplegic paralysis (7 patients, 70%), dizziness (5 patients, 50%), and unclear speech (3 patients, 30%). The median age was 63.5 years. Eight and two cases were stage III and IV, respectively, at the initial diagnosis. Five patients underwent driver gene testing, revealing three patients with EGFR-sensitive mutations, one patient with ALK fusion, and one patient with wild-type EGFR. All 10 patients received antiplatelet therapy, and six patients subsequently received anti-cancer treatment. The median overall survival of the patients was 8.5 months (95% confidence interval) and 1-year survival rate was 57.1%. Patients who received antitumor treatment, especially those harboring driver gene mutations and received tyrosine kinase inhibitors, had better neurological symptom recovery and superior oncological prognosis (median overall survival, not reached versus 7.4 months, p = 0.038). CONCLUSION: Trousseau syndrome, presenting as multiple cerebral infarctions, is a rare complication of lung adenocarcinoma. Both antiplatelet and antitumor treatment are recommended to achieve better neurological recovery and oncological prognosis in these patients.

Partial Ablation of Astrocytes Exacerbates Cerebral Infiltration of Monocytes and Neuronal Loss After Brain Stab Injury in Mice.

In traumatic brain injury (TBI), mechanical injury results in instantaneous tissue damages accompanied by subsequent pro-inflammatory cascades composed of microgliosis and astrogliosis. However, the interactive roles between microglia and astrocytes during the pathogenesis of TBI remain unclear and sometimes debatable. In this study, we used a forebrain stab injury mouse model to investigate the pathological role of reactive astrocytes in cellular and molecular changes of inflammatory response following TBI. In the ipsilateral hemisphere of stab-injured brain, monocyte infiltration and neuronal loss, as well as increased elevated astrogliosis, microglia activation and inflammatory cytokines were observed. To verify the role of reactive astrocytes in TBI, local and partial ablation of astrocytes was achieved by stereotactic injection of diphtheria toxin in the forebrain of Aldh1l1-CreERT2::Ai9::iDTR transgenic mice which expressed diphtheria toxin receptor (DTR) in astrocytes after tamoxifen induction. This strategy achieved about 20% of astrocytes reduction at the stab site as validated by immunofluorescence co-staining of GFAP with tdTomato-positive astrocytes. Interestingly, reduction of astrocytes showed increased microglia activation and monocyte infiltration, accompanied with increased severity in stab injury-induced neuronal loss when compared with DTR-/- mice, together with elevation of inflammatory chemokines such as CCL2, CCL5 and CXCL10 in astrogliosis-reduced mice. Collectively, our data verified the interactive role of astrocytes as an immune modulator in suppressing inflammatory responses in the injured brain. Schematic diagram shows monocyte infiltration and neuronal loss, as well as increased elevated astrogliosis, microglia activation and chemokines were observed in the injured site after stab injury. Local and partial ablation of astrocytes led to increased microglia activation and monocyte infiltration, accompanied with increased severity in neuronal loss together with elevation of inflammatory chemokines as compared with control mice subjected stab injury.

The shape effect and its consequences for polar surfaces and for heterogeneous catalysis.

In this paper we develop the shape effect, which is relevant for crystalline materials whose size is larger than that of the thermodynamic limit. According to this effect the electronic properties of one surface of a crystal depend upon all of its surfaces, i.e. on the overall shape. At first, qualitative mathematical arguments are presented for the existence of this effect based on the conditions for the stability of polar surfaces. Our treatment explains why such surfaces are observed even though earlier theory indicated that they should not exist. Then, models are developed from which it is found computationally that changing the shape of a polar crystal can substantially alter the magnitude of its surface charges. Apart from surface charges, it follows that the crystal shape will also significantly affect bulk properties, most notably polarization and piezoelectric responses. Additional model calculations show a strong shape effect on the activation energy for heterogeneous catalysis primarily through local surface charges rather than a non-local/long range electrostatic potential.

Effect of peroxiredoxin 1 on the regulation of trophoblast function by affecting autophagy and oxidative stress in preeclampsia.

PURPOSE: PE is a pregnancy-specific syndrome and one of the main causes of maternal, fetal, and neonatal mortality. PRDX1 is an antioxidant that regulates cell proliferation, differentiation, and apoptosis. The aim of this study is to investigate the effect of PRDX1 on the regulation of trophoblast function by affecting autophagy and oxidative stress in preeclampsia. METHODS: Western blotting, RT-qPCR, and immunofluorescence were used to examine the expression of PRDX1 in placentas. PRDX1-siRNA was transfected to knockdown PRDX1 in HTR-8/SVneo cells. The biological function of HTR-8/SVneo cells was detected by wound healing, invasion, tube formation, CCK-8, EdU, flow cytometry, and TUNEL assays. Western blotting was used to detect the protein expression of cleaved-Caspase3, Bax, LC3II, Beclin1, PTEN, and p-AKT. DCFH-DA staining was used to detect ROS levels by flow cytometry. RESULTS: PRDX1 was significantly decreased in placental trophoblasts in PE patients. Following the exposure of HTR-8/SVneo cells to H2O2, PRDX1 expression was significantly decreased, LC3II and Beclin1 expression was notably increased, and ROS level was also markedly increased. PRDX1 knockdown impaired migration, invasion, and tube-formation abilities and promoted apoptosis, which was accompanied by an increased expression of cleaved-Caspase3 and Bax. PRDX1 knockdown induced a significant decrease in LC3II and Beclin1 expression, along with an elevated p-AKT expression and a decreased PTEN expression. PRDX1 knockdown increased intracellular ROS levels, and NAC attenuated PRDX1 knockdown-induced apoptosis. CONCLUSION: PRDX1 regulated trophoblast function through the PTEN/AKT signaling pathway to affect cell autophagy and ROS level, which provided a potential target for the treatment of PE.

Bacterial O-GlcNAcase genes abundance decreases in ulcerative colitis patients and its administration ameliorates colitis in mice.

OBJECTIVE: O-linked N-acetylglucosaminylation (O-GlcNAcylation), controlled by O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), is an important post-translational modification of eukaryotic proteins and plays an essential role in regulating gut inflammation. Gut microbiota encode various enzymes involved in O-GlcNAcylation. However, the characteristics, abundance and function of these enzymes are unknown. DESIGN: We first investigated the structure and taxonomic distribution of bacterial OGAs and OGTs. Then, we performed metagenomic analysis to explore the OGA genes abundance in health samples and different diseases. Finally, we employed in vitro and in vivo experiments to determine the effects and mechanisms of bacterial OGAs to hydrolyse O-GlcNAcylated proteins in host cells and suppress inflammatory response in the gut. RESULTS: We found OGAs, instead of OGTs, are enriched in Bacteroidetes and Firmicutes, the major bacterial divisions in the human gut. Most bacterial OGAs are secreted enzymes with the same conserved catalytic domain as human OGAs. A pooled analysis on 1999 metagenomic samples encompassed six diseases revealed that bacterial OGA genes were conserved in healthy human gut with high abundance, and reduced exclusively in ulcerative colitis. In vitro studies showed that bacterial OGAs could hydrolyse O-GlcNAcylated proteins in host cells, including O-GlcNAcylated NF-κB-p65 subunit, which is important for activating NF-κB signalling. In vivo studies demonstrated that gut bacteria-derived OGAs could protect mice from chemically induced colonic inflammation through hydrolysing O-GlcNAcylated proteins. CONCLUSION: Our results reveal a previously unrecognised enzymatic activity by which gut microbiota influence intestinal physiology and highlight bacterial OGAs as a promising therapeutic strategy in colonic inflammation.

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung.

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Long noncoding RNA lnc-RI regulates DNA damage repair and radiation sensitivity of CRC cells through NHEJ pathway.

A percentage of colorectal cancer (CRC) patients display low sensitivity to radiotherapy, which affects its therapeutic effect. Cancer cells DNA double-strand breaks (DSBs) repair capacity is crucial for radiosensitivity, but the roles of long noncoding RNAs (lncRNAs) in this process are largely uncharacterized. This study aims to explore whether lnc-RI regulates CRC cell growth and radiosensitivity by regulating the nonhomologous end-joining (NHEJ) repair pathway. CRC cells in which lnc-RI has been silenced showed lower cell growth and higher apoptosis rates due to increased DSBs and cell cycle arrest. We found that miR-4727-5p targets both lnc-RI and LIG4 mRNA and inhibit their expression. CRC cells showed increased radiosensitivity when lnc-RI was silenced. These results reveal novel roles for lnc-RI in both DNA damage repair and radiosensitivity regulation in CRC cells. Our study revealed that lnc-RI regulates LIG4 expression through lnc-RI/miR-4727-5p/LIG4 axis and regulates NHEJ repair efficiency to participate in DNA damage repair. The level of lnc-RI was negatively correlated with the radiosensitivity of CRC cells, indicates that lnc-RI may be a potential target for CRC therapy. We also present the first report of the function of miR-4727-5p.

Theoretical study of the mechanism behind the site- and enantio-selectivity of C-H functionalization catalysed by chiral dirhodium catalyst.

The C-H functionalization is very important for the synthesis of pharmaceuticals and complex natural products. Rhodium carbenoids, obtained when a dirhodium(ii) catalyst containing a crown formed by chiral ligands reacts with diazo compounds with both an electron donating group and an electron withdrawing group, play an important part in controlling site- and enantio-selectivity for functionalization of non-activated C-H bonds. It has earlier been demonstrated that the tertiary C-H bond is more favored to be functionalized inside the crown of the dirhodium catalyst with S-configuration ligands compared with the secondary and primary C-H bonds although the latter possess weaker steric effects. We argue that the higher site- and enantio-selectivity for some types of C-H bond functionalization can be related to intermolecular hydrogen bonding, steric hindrance, and weak interactions when the dirhodium catalyst is interacting with the chiral ligands.

STK33 alleviates gentamicin-induced ototoxicity in cochlear hair cells and House Ear Institute-Organ of Corti 1 cells.

Serine/threonine kinase 33 (STK33), a member of the calcium/calmodulin-dependent kinase (CAMK), plays vital roles in a wide spectrum of cell processes. The present study was designed to investigate whether STK33 expressed in the mammalian cochlea and, if so, what effect STK33 exerted on aminoglycoside-induced ototoxicity in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. Immunofluorescence staining and western blotting were performed to investigate STK33 expression in cochlear hair cells (HCs) and HEI-OC1 cells with or without gentamicin treatment. CCK8, flow cytometry, immunofluorescence staining and western blotting were employed to detect the effects of STK33 knockdown, and/or U0126, and/or N-acetyl-L-cysteine (NAC) on the sensitivity to gentamicin-induced ototoxicity in HEI-OC1 cells. We found that STK33 was expressed in both mice cochlear HCs and HEI-OC1 cells, and the expression of STK33 was significantly decreased in cochlear HCs and HEI-OC1 cells after gentamicin exposure. STK33 knockdown resulted in an increase in the cleaved caspase-3 and Bax expressions as well as cell apoptosis after gentamicin damage in HEI-OC1 cells. Mechanistic studies revealed that knockdown of STK33 led to activated mitochondrial apoptosis pathway as well as augmented reactive oxygen species (ROS) accumulation after gentamicin damage. Moreover, STK33 was involved in extracellular signal-regulated kinase 1/2 pathway in primary culture of HCs and HEI-OC1 cells in response to gentamicin insult. The findings from this work indicate that STK33 decreases the sensitivity to the apoptosis dependent on mitochondrial apoptotic pathway by regulating ROS generation after gentamicin treatment, which provides a new potential target for protection from the aminoglycoside-induced ototoxicity.