primary human corneal epithelial cells hcecs  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of miR-16-5p and miR-142-3p on Inflammation and Autophagy in Human Corneal Epithelial Cells Under Hyperosmotic Stress In Vitro

doi: 10.3390/ijms27010422

Figure Lengend Snippet: Establishment and molecular validation of a hyperosmolar stress model in human corneal epithelial (hCE) cells. ( A ) Representative phase-contrast morphology of HCEpiCs under iso-osmotic control and hyperosmotic stress (450 mOsm NaCl, 24 h). Scale bar = 100 μm. ( B ) Cell viability analysis (CCK-8) revealed a dose- and time-dependent reduction, with ~30% viability loss at 450 mOsm for 24 h, selected as the optimal condition. ( C ) Western blot analysis demonstrated induction of inflammatory mediators (IL-1β, IL-6, TNF-α, IRAK1). ( D ) Hyperosmotic stress upregulated autophagy-related proteins (ATG5, Beclin-1, ATG16L1) and altered p62 expression, indicating autophagic activation. GAPDH served as the loading control, and all protein levels were normalized to GAPDH. ( E ) Apoptotic signaling was evident with Bax upregulation, Bcl-2 suppression, and increased cleaved caspase-3. * p < 0.05, ** p < 0.01 versus control.

Article Snippet: Human corneal epithelial cells (HCEpiCs) were purchased from Innoprot (Derio, Bizkaia, Spain) and maintained in Corneal Epithelial Cell Medium (Innoprot, Derio, Bizkaia, Spain) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Biomarker Discovery, Control, CCK-8 Assay, Western Blot, Expressing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of miR-16-5p and miR-142-3p on Inflammation and Autophagy in Human Corneal Epithelial Cells Under Hyperosmotic Stress In Vitro

doi: 10.3390/ijms27010422

Figure Lengend Snippet: miR-16-5p and miR-142-3p repress hyperosmolarity-induced inflammatory signaling. ( A ) qPCR analysis of IL-1β, IL-6, and TNF-α expression in HCEpiCs under hyperosmotic stress with or without miR-16-5p/miR-142-3p mimic transfection. Data are presented as mean ± SEM. Each dot represents an individual biological replicate. ( B ) Western blot analysis and densitometric quantification showing that both miRNAs significantly attenuated cytokine induction (IL-1β, IL-6, TNF-α, IRAK1). GAPDH served as the loading control, and all protein levels were normalized to GAPDH. * p < 0.05, *** p < 0.001 versus control. # p < 0.05, ## p < 0.01, ### p < 0.001 versus NaCl group.

Article Snippet: Human corneal epithelial cells (HCEpiCs) were purchased from Innoprot (Derio, Bizkaia, Spain) and maintained in Corneal Epithelial Cell Medium (Innoprot, Derio, Bizkaia, Spain) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Expressing, Transfection, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of miR-16-5p and miR-142-3p on Inflammation and Autophagy in Human Corneal Epithelial Cells Under Hyperosmotic Stress In Vitro

doi: 10.3390/ijms27010422

Figure Lengend Snippet: Autophagy regulation by miR-16-5p and miR-142-3p in HCEpiCs. ( A ) qPCR analysis of autophagy-related genes (ATG5, Beclin-1, ATG16L1, p62) in hyperosmotic stress ± miRNA mimics. Data are presented as mean ± SEM. Each dot represents an individual biological replicate. ( B ) Western blot analysis and quantification of autophagy proteins (ATG5, Beclin-1, ATG16L1, p62). GAPDH served as the loading control, and all protein levels were normalized to GAPDH. ( C ) Representative DAPRed fluorescence imaging of autophagic vesicles (red) and quantitative analysis of fluorescence intensity. Both miRNAs reduced autophagy marker expression and vesicle accumulation under hyperosmotic conditions. * p < 0.05, *** p < 0.001 versus control. # p < 0.05, ## p < 0.01, ### p < 0.001 versus NaCl group.

Article Snippet: Human corneal epithelial cells (HCEpiCs) were purchased from Innoprot (Derio, Bizkaia, Spain) and maintained in Corneal Epithelial Cell Medium (Innoprot, Derio, Bizkaia, Spain) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Western Blot, Control, Fluorescence, Imaging, Marker, Expressing

Journal: EMBO Molecular Medicine

Article Title: Corneal biomechanical cues mediated by PAI-2: the origin of PM2.5-induced corneal disease

doi: 10.1038/s44321-025-00341-0

Figure Lengend Snippet: ( A ) Immunofluorescence staining for phosphorylated non-muscle myosin IIa of HCECs exposed for different times to PM2.5 (scale bar, 75 μm) ( n = 3 biological replicates). ( B ) Cellular tensile stress of HCECs exposed to PM2.5 (with or without blebb) for 3 h was measured by traction force microscopy ( n = 40 for control, n = 32 for PM, n = 25 for PM+blebb). ( C ) Cellular stiffness of HCECs exposed to PM2.5 for 3 h measured by atomic force microscopy ( n = 8). ( D ) Cellular viability of HECEs in the presence of RI or blebb ( n = 3 biological replicates). ( E ) Impression cytology was used to evaluate rat corneal epithelial cell circularity in two groups (scale bar, 25 μm). n = 5 (PBS), 9 (PM). ( F ) PM2.5 exposure leads to the activated biomechanical response at the subclinical stage in humans, rats, and HCECs. This curve provides a conceptual illustration of the correlation between biomechanical response and PM2.5-induced corneal disease progression; it does not reflect precise numerical changes. Data in ( A – E ) are graphed as mean ± standard deviation with individual values shown as dots or circles. Statistical analysis was conducted using the unpaired t test in ( A – E ). The P values are labeled in the figure. .

Article Snippet: Human corneal epithelial cell lines , ATCC , PCS-700-010.

Techniques: Immunofluorescence, Staining, Microscopy, Control, Biomarker Discovery, Standard Deviation, Labeling

Journal: EMBO Molecular Medicine

Article Title: Corneal biomechanical cues mediated by PAI-2: the origin of PM2.5-induced corneal disease

doi: 10.1038/s44321-025-00341-0

Figure Lengend Snippet: ( A ) PAI-2 staining in rat cornea after short-term (2-day) PM2.5 exposure (scale bar, 25 μm). The while arrow refers to the rat corneal superficial epithelial cells. n = 4. ( B ) The mRNA level of PAI-2 in rat corneas after short-term (2-day) PM2.5 exposure. n = 4. ( C ) The protein level of PAI-2 in HCECs after short-term (3-h) PM2.5 exposure ( n = 3 biological replicates). ( D ) The viability of NC and KO after 24-h PM2.5 treatment ( n = 3 biological replicates). ( E ) The heatmap showed the different expression profiles in three groups. ( F ) The Venn diagram demonstrated the common DEGs between the two sets of comparisons. ( G ) GO analysis revealed biological process, cellular component, and molecular function enriched in DEGs. ( H ) KEGG pathway analysis of DEGs. ( I ) PAI-2 is associated with several cellular mechanics-related processes, which may regulate PM2.5-induced cellular mechanical response. Data in ( A – D ) are graphed as mean ± standard deviation with individual values shown as circles. Statistical analysis was conducted using the unpaired t test in ( A – D ). The P values are labeled in the figure. .

Article Snippet: Human corneal epithelial cell lines , ATCC , PCS-700-010.

Techniques: Staining, Expressing, Standard Deviation, Labeling

Journal: EMBO Molecular Medicine

Article Title: Corneal biomechanical cues mediated by PAI-2: the origin of PM2.5-induced corneal disease

doi: 10.1038/s44321-025-00341-0

Figure Lengend Snippet: ( A ) The workflow of extracellular PAI-2 detection in rat tears, HCEC conditioned medium, and human tears. For the details of the panel study in humans, participants completed 24 h of continuous personal air monitoring followed by a questionnaire survey, tear sample collection, and ophthalmic examination. Tear samples were then sent to a laboratory to detect PAI-2 content. n = 28. ( B ) The PAI-2 concentration in rat tears upon PM2.5 exposure for different times was detected via ELISA. n = 5 (PBS group), 6 (PM2.5 group). ( C ) The PAI-2 level in HCEC conditioned medium upon 24-h PM2.5 exposure was detected via ELISA ( n = 7 biological replicates). ( D – I ) Spearman analysis was performed to separately define the correlation between PM2.5 concentration and ocular surface disease index/eyesight/tear film break-up time/tear secretion/corneal epithelial defect/PAI-2 content in tears. n = 28. Individual values are shown as scattered dots. ( J ) PIPs of PM2.5 constituents on the PAI-2 level calculated by the BKMR model. ( K ) Univariate exposure−response curves for PM2.5 constituents with PIPs above 0.9, while all other PM2.5 elemental constituents were at their median concentrations (shown with credible interval). Data in ( B , C ) are graphed as mean ± standard deviation with individual values shown as circles. Statistical analysis was conducted using the unpaired t test in ( B , C ). The P values are labeled in the figure. .

Article Snippet: Human corneal epithelial cell lines , ATCC , PCS-700-010.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Labeling

Journal: EMBO Molecular Medicine

Article Title: Corneal biomechanical cues mediated by PAI-2: the origin of PM2.5-induced corneal disease

doi: 10.1038/s44321-025-00341-0

Figure Lengend Snippet: ( A ) The corneal epithelium defect was examined by slit lamp examination using fluorescein sodium staining under cobalt-blue light. The red arrows pointed to the corneal epithelium defect. The fluorescein sodium staining grades were evaluated. n = 6. ( B ) H&E staining showed the morphological changes in the rat cornea, lens, and retina with different treatments (scale bar, 50 μm). The corneal epithelial thickness was measured based on H&E staining. n = 4 (PBS), 4 (PM), 4 (PM + LNP), 3 (PM + LNP-siNC), 4 (PM + LNP-siPAI-2). ( C ) Immunofluorescence staining of rat cornea for PAI-2 and LC3B (scale bar, 50 μm). n = 3. The fluorescence intensities of PAI-2 and LC3B were calculated based on immunofluorescence staining. ( D ) Immunohistochemistry showed the expression level of IL-1a of rat corneas in different groups (scale bar, 50 μm). n = 4 (PBS), 3 (PM), 3 (PM + LNP), 3 (PM + LNP-siNC), 4 (PM + LNP-siPAI-2). The average optical density of IL-1a in rat corneal epithelium was measured based on immunohistochemistry. ( E ) The rat model workflow and therapeutic mechanism diagram of the LNP-siPAI-2 system. LNP-siPAI-2 delivers siPAI-2 to rat corneal epithelial cells, thereby targeting and reducing PAI-2 expression levels and inhibiting autophagy and IL-1a to attenuate corneal epithelial cell damage. At the same time, the reduction in corneal damage reduces the inflammatory response of the intraocular lens and retina, thus realizing the protection of the whole eye by the LNP-siPAI-2 system. Data in ( A – D ) are graphed as mean ± standard deviation with individual values shown as circles, squares, or triangles. Statistical analysis was conducted using the unpaired t test in ( A – D ). The P values are labeled in the figure. .

Article Snippet: Human corneal epithelial cell lines , ATCC , PCS-700-010.

Techniques: Staining, Immunofluorescence, Fluorescence, Immunohistochemistry, Expressing, Standard Deviation, Labeling

Journal: EMBO Molecular Medicine

Article Title: Corneal biomechanical cues mediated by PAI-2: the origin of PM2.5-induced corneal disease

doi: 10.1038/s44321-025-00341-0

Figure Lengend Snippet: ( A ) DLin-MC3-DMA LNPs can deliver the load (luciferase mRNA) into the corneal epithelium of rats (scale bar, 75 μm). n = 4. ( B ) Detection of PAI-2 knockdown efficiency by LNP (siPAI-2) ( n = 3 biological replicates). ( C ) Immunohistochemistry showed the expression level of IL-1a in rat lens and retinas of different groups (scale bar, 50 μm). n = 4 (PBS), 3 (PM), 3 (PM + LNP), 3 (PM + LNP-siNC), 4 (PM + LNP-siPAI-2). The red boxed refer to the rat lens epithelial cells. The average optical density of IL-1a in rat lens and retinas of different groups. ( D ) The retinal thickness based on H&E staining in Fig. . n = 4 (PBS), 3 (PM), 4 (PM + LNP), 3 (PM + LNP-siNC), 4 (PM + LNP-siPAI-2). Data in ( A – D ) are graphed as mean ± standard deviation with individual values shown as circles, squares, or triangles. Statistical analysis was conducted using the unpaired t test in ( A – D ). The P values are labeled in the figure.

Article Snippet: Human corneal epithelial cell lines , ATCC , PCS-700-010.

Techniques: Luciferase, Knockdown, Immunohistochemistry, Expressing, Staining, Standard Deviation, Labeling