mle12 cell line  (Corning Life Sciences)

Journal: bioRxiv

Article Title: Cryptochrome Loss Drives COPD-like Lung Pathology through Disrupted Alveolar Epithelial Proliferation and Immune Homeostasis

doi: 10.64898/2026.05.19.726266

Figure Lengend Snippet: MLE12 cells were transfected with 50 pmole/ml control, Cry1 , or Cry2 siRNA on day 0 and day 1. A. Western blot showed CRY1/CRY2 protein levels on day 2. B. The transcript levels of cytokines, including C-C motif chemokine ligand 2 ( Ccl2 ), C-X-C motif chemokine ligand 1 ( Cxcl1 ), Interleukin-6 ( Il6 ), Il8/Cxcl5 , matrix metalloproteinase 1 and 7 ( Mmp1 and Mmp7 ), and Tnfa were determined with RT-qPCR. C. Cell numbers were measured with the WST1 assay 1-, 2-, or 3-days post siRNA transfection. n=6. D-E. Primary alveolar ECs were isolated from WT or Cry1/2 dKO mice for RNA sequencing analysis. D. Heatmap image of up (501) and down (369) regulated genes in Cry1/2 dKO mice alveolar ECs compared to WT alveolar ECs. Each gene is represented as a horizontal line. E. GO Biological Process (BP) analysis of DEGs that were in Cry1/2 dKO lung ECs. F. GO Molecular Function (MF) analysis of DEGs. G. GO Cellular Component (CC) analysis of DEGs. The top 5 terms were shown for both up and down-regulated genes. H. Enriched pathways (Metascape) of overlapping genes from human COPD patient ECs DEGs with DEGs from (D). I. GO BP, J. MF and K . CC analysis of common genes between human COPD patient lungs with DEGs in Cry1/2 dKO ECs. Data are presented as mean ± SEM. ** p<0.1, *** p<0.001, and **** p<0.0001 Cry1/2 si vs si_con (Two-way ANOVA followed by Bonferroni adjusted multiple comparisons). For C , only the asterisks for si- Cry1/2 are shown.

Article Snippet: Human lung epithelial cell line A549 and mouse primary lung epithelial cell line MLE12 were procured from ATCC (Manassas, VA).

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: Cryptochrome Loss Drives COPD-like Lung Pathology through Disrupted Alveolar Epithelial Proliferation and Immune Homeostasis

doi: 10.64898/2026.05.19.726266

Figure Lengend Snippet: A. Experimental schematic: Cry1/2 dKO mice were fed with regular (RD) or 1% NOB-containing diets starting at 1 month old and analyzed at 4 months. B. Representative images of H&E-stained lung sections collected from mice fed with a RD or NOB diet. C. MLI quantification of H&E-stained lung sections from male or female Cry1/2 dKO fed with RD or NOB diets. D. RNA-seq was performed using primary lung ECs isolated from the WT mice, or Cry1/2 dKO mice fed with RD or NOB diet. Heatmap of RNA-seq data of lung ECs from different treatment groups. E. GO BP analysis of DEGs that were rescued by NOB treatment in Cry1/2 dKO lung ECs. F. GO MF analysis of rescued DEGs. G. GO CC analysis of rescued DEGs. H. RT qPCR confirmed the upregulation and rescue of genes involved in cilium movement. I. The transcript expression changes of important differentially expressed chemokines, cytokines, Mmps , and surfactant proteins from RNA-seq data. Data were presented as fold changes to WT. J. MlE12 cells were transfected with control siRNA or siRNA targeting Cry1 and Cry2 . 36 h after transfection, cells were treated with 5 ng/ml TNF-α for 15 min. Phosphorylated P-65 and P65 levels were determined with Western blots. K. MLE12 cells were treated with 20 µM NOB for 24 h, followed by 5 ng/ml TNF-α for 0, 15, or 30 mins. Phosphorylated P-65 and P65 levels were determined with Western blots. L. MLE12 cells were transfected with control siRNA, or siRNA targeting Cry1 and Cry2 , and treated with 20 µM NOB for 24 h before TNF-α stimulation (15 mins). Representative confocal images of p65 (green) are shown (×400 magnification; scale bar = 10 µm). M. The percentage of cells with p65 localized in the cytoplasm (C), both cytoplasm and nucleus (C+N), or nucleus (N) was quantified for each treatment condition. N. Western blot of CRY1/2 and COPD-associated proteins in primary lung ECs from different treatment groups. Data are presented as mean ± SEM. P-value was calculated from a two-tailed t-test for H and I, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 in RD vs in Cry1/2 dKO samples.

Article Snippet: Human lung epithelial cell line A549 and mouse primary lung epithelial cell line MLE12 were procured from ATCC (Manassas, VA).

Techniques: Staining, RNA Sequencing, Isolation, Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Two Tailed Test

Journal: Inflammation

Article Title: Inhibition of TRPV4 Regulates Mitophagy Through the Sirt1/FoxO1 Signaling Pathway To Alleviate Acute Lung Injury

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: Inhibition of TRPV4 increased mitochondrial autophagy and attenuated damage of MLE12 cells. A CCK-8 assay was detected to evaluate cell viability in each group ( n = 6) B , C Intracellular calcium ion concentrations were measured using laser confocal microscopy (original magnification,×64; scale bar: 50 μm) D , E Fluorescence microscopy was used to assess ROS levels F Cell apoptosis in each group was detected by flow cytometry (original magnification,×64; scale bar: 50 μm) ( n = 6) G , H Changes in mitochondrial membrane potential were detected using laser confocal microscopy with JC-1 staining(original magnification, ×64; scale bar: 50 μm) (×64; scale bar: 20 μm) ( n = 6). Nuclei were revealed using DAPI staining I The expressions of autophagy-related proteins PINK1 and PARK2 and their upstream regulatory factor Sirt1、FoxO1 in mitochondria were measured by Western Blot.

Article Snippet: The mouse lung epithelial cell line MLE12 was bought from American Type Culture Collection (ATCC, CRL-2110, USA).

Techniques: Inhibition, CCK-8 Assay, Confocal Microscopy, Fluorescence, Microscopy, Flow Cytometry, Membrane, Staining, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway

doi: 10.1186/s12964-024-01946-7

Figure Lengend Snippet: Pyroptosis is the primary mode of AEC death induced by cadmium. MLE12 cells were treated with different concentrations of CdCl 2 for 24 h. Cell morphology was photographed through a microscope (A, bar = 100 μm). Cell viability was detected by CCK8 assay (B, n = 5). The LDH activity of the cell culture supernatant was assayed (C, n = 3). The Pan-caspase inhibitor (ZVAD-FMK) but not the necroptosis or ferroptosis inhibitor restored cell viability within 24 h of CdCl 2 incubation (D, n = 5). Cell morphology was photographed through a microscope (E, bar = 100 μm). The expressions of SPC, NLRP3, pro-Caspase1, Caspase1 p20, IL-1β p17, and GSDMD-N protein in MLE12 cells were detected using western blot (F-G, n = 3). The fluorescence intensity of GSDMD was detected by immunofluorescence (H, bar = 5 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Mouse alveolar epithelial cell lines MLE12 were purchased from ATCC (USA, CRL-2110).

Techniques: Microscopy, CCK-8 Assay, Activity Assay, Cell Culture, Incubation, Western Blot, Fluorescence, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway

doi: 10.1186/s12964-024-01946-7

Figure Lengend Snippet: Cadmium activates the cGAS-STING pathway in AECs. Differentially expressed genes in the MLE12 cells of Control and CdCl 2 groups ( n = 3). PCA analysis (A). Volcano plot showing the changes of MLE12 genes (fold change ≥ 2) (B), GO enrichment analysis for differentially expressed genes (C). KEGG enrichment analysis for differentially expressed genes (D). The gene expression levels of the cGAS-STING pathway (E, n = 3). The expressions of cGAS and STING protein in the lung were detected using western blot (F-G, n = 3). The expressions of cGAS and STING protein and the phosphorylation level of TBK1 in MLE12 cells were detected using western blot (H-I, n = 3). The fluorescence intensity of STING was detected by immunofluorescence (J, bar = 5 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * P < 0.05 and *** P < 0.001

Article Snippet: Mouse alveolar epithelial cell lines MLE12 were purchased from ATCC (USA, CRL-2110).

Techniques: Control, Gene Expression, Western Blot, Phospho-proteomics, Fluorescence, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway

doi: 10.1186/s12964-024-01946-7

Figure Lengend Snippet: Cadmium exposure induces pyroptosis in AECs by activating the cGAS-STING pathway. MLE12 cells were treated with CdCl 2 for 24 h after RU.521 intervention for 30 min. The expression of STING protein and the phosphorylation level of TBK1 and NF-κB p65 in MLE12 cells were detected using Western blot (A-B, n = 3). The expression of pro-Il-1β mRNA in MLE12 cells was detected using Real-time PCR (C, n = 3). The expressions of NLRP3, pro-Caspase1, Caspase1 p20, pro-IL-1β, IL-1β p17, and GSDMD protein in MLE12 cells were detected using western blot (D-E, n = 3). The fluorescence intensity of GSDMD was detected by immunofluorescence (F, bar = 5 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Mouse alveolar epithelial cell lines MLE12 were purchased from ATCC (USA, CRL-2110).

Techniques: Expressing, Phospho-proteomics, Western Blot, Real-time Polymerase Chain Reaction, Fluorescence, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway

doi: 10.1186/s12964-024-01946-7

Figure Lengend Snippet: Mitochondrial oxidative stress induced by cadmium exposure activates the cGAS-STING pathway to induce pyroptosis in AECs. MLE12 cells were treated with CdCl 2 for 24 h after mitoQ intervention for 30 min. Mitochondrial membrane potential was measured by JC-1 staining (A, bar = 50 μm). ROS and mitoROS were detected by fluorescent probes (B, bar = 100 μm). Cytosolic mtDNA ND1 and Dloop2 levels in MLE12 (C, n = 3). The expressions of pro-Il-1β and Gsdmd mRNA in MLE12 cells were detected using Real-time PCR (D, n = 3). The expressions of NLRP3, Caspase-1 p20, IL-1β p17, GSDMD, and STING protein and the phosphorylation level of TBK1 and NF-κB p65 in MLE12 cells were detected using western blot (E–F, n = 3). The fluorescence intensity of STING was detected by immunofluorescence (G, bar = 5 μm). The fluorescence intensity of GSDMD was detected by immunofluorescence (H, bar = 5 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Mouse alveolar epithelial cell lines MLE12 were purchased from ATCC (USA, CRL-2110).

Techniques: Membrane, Staining, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Fluorescence, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway

doi: 10.1186/s12964-024-01946-7

Figure Lengend Snippet: Excessive accumulation of cytoplasmic mtDNA activates the cGAS-STING pathway to induce pyroptosis in cadmium-exposed AECs. MLE12 cells were treated with CdCl 2 for 24 h after VBIT4 intervention for 30 min. Cytosolic mtDNA ND1 and Dloop2 levels in MLE12 (A, n = 3). The expressions of IL-1β p17, GSDMD, cGAS, and STING protein and the phosphorylation level of TBK1 and NF-κB p65 in MLE12 cells were detected using western blot (B-C, n = 3). The fluorescence intensity of STING was detected by immunofluorescence (D, bar = 10 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Mouse alveolar epithelial cell lines MLE12 were purchased from ATCC (USA, CRL-2110).

Techniques: Phospho-proteomics, Western Blot, Fluorescence, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway

doi: 10.1186/s12964-024-01946-7

Figure Lengend Snippet: Inhibition of the cGAS-STING pathway reduces lung injury and AEC pyroptosis induced by cadmium in mice. C57BL/6 J mice were given a daily intraperitoneal injection of CdCl 2 (3 mg/kg) and a daily intraperitoneal injection of RU.521 (5 mg/kg) (A). Weight change of mice (B, n = 7–10). The appearance of lung tissue (C). HE staining of lung tissue (D, bar = 100 μm). The inflammatory injury score was performed in a double-blind fashion (E, n = 6). The expressions of Nlrp3, pro-Il-1β, pro-Caspase1, Gsdmd, Sting, and Tbk1 mRNA in MLE12 cells were detected using Real-time PCR (F, n = 3). The expressions of NLRP3, Caspase-1 p20, IL-1β p17, GSDMD-N, cGAS, and STING protein and the phosphorylation level of TBK1 in the lung were detected using western blot (G-H, n = 3). The fluorescence intensity of SFTPC and GSDMD was detected by immunofluorescence (I, bar = 100 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Mouse alveolar epithelial cell lines MLE12 were purchased from ATCC (USA, CRL-2110).

Techniques: Inhibition, Injection, Staining, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Fluorescence, Immunofluorescence