mouse primary lung epithelial cell line mle12  (ATCC)

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: Scientific reports

Article Title: CPT1A as a potential therapeutic target for lipopolysaccharide-induced acute lung injury in mice.

doi: 10.1038/s41598-024-52042-2

Figure Lengend Snippet: Figure 4. Effects of CPT1A on LPS-induced apoptosis in MLE12 cells in vitro. (A) Caspase-3 activity in MLE12 cells treated with Lenti-CPT1A was determined. (B, C) The apoptosis rates of MLE12 cells treated with Lenti-CPT1A were determined by flow cytometry. (D, E) Bcl-2 and Bax mRNA in MLE12 cells treated with Lenti-CPT1A were determined. (F–H) BCL-2 and BAX protein in MLE12 cells treated with Lenti-CPT1A were determined. (I) Caspase-3 activity in MLE12 cells treated with etomoxir (10 μM) was determined. (J-K) Bcl-2 and Bax mRNA in MLE12 cells treated with etomoxir (10 μM) were determined. n = 4, *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Mouse lung epithelial cell line MLE12 cells were obtained from American Type Culture Collection (ATCC).

Techniques: In Vitro, Activity Assay, Flow Cytometry

Journal: Scientific reports

Article Title: CPT1A as a potential therapeutic target for lipopolysaccharide-induced acute lung injury in mice.

doi: 10.1038/s41598-024-52042-2

Figure Lengend Snippet: Figure 5. CPT1A overexpression mitigated mitochondrial dysfunction and restored FAO in lung tissue of ALI mice and LPS-treated MLE12 cells. (A) Mitochondrial DNA copy number (mtDNA) was determined in the lungs. (B) Radiolabeled palmitate-derived CO2 was determined after incubating 14C-palmitate with lung tissue after doxycycline induction. (C) ATP levels in total lung tissue. (D) Peroxisomal/mitochondrial function-associated genes were determined in the lungs after doxycycline induction. (E) OCR of MLE12 cells was measured with a Seahorse XF24 Extracellular Flux Analyzer. Bar graphs show the rates of OCR associated with basal, proton-leak, ATP-linked, maximum, reserve capacity, and nonmitochondrial respiratory statuses. (F) Extracellular acidification rate (ECAR) of MLE12 cells. (G) ATP levels of MLE12 cells. n = 6, *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Mouse lung epithelial cell line MLE12 cells were obtained from American Type Culture Collection (ATCC).

Techniques: Over Expression, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

doi: 10.4049/jimmunol.2200245

Figure Lengend Snippet: FIGURE 5. FBXL2 ubiquitinates and regulates T-bet. (A) In vitro ubiquitination assay using puri- fied E1/E2/E3 complex components incubated with T-betHA and the full complement of ubiqui- tination reaction components (second lane from left). Data representative of three replicate experi- ments. Jurkat cells were stimulated with/without anti-CD3/CD28 Abs for 24 h, and cells were then lysed and either T-bet (B) or FBXL2 (C) was immunoprecipitated (IP) followed by FBXL2 (B) or T-bet (C) immunoblotting. Data are representa- tive of three replicate experiments. (D) MLE12 cells were transfected with increasing amounts of FBXL2 E3 ligase plasmids with a fixed amount of T-bet plasmid for 18 h. The cell lysates were sepa- rated by SDS-PAGE (bottom) and analyzed by immunoblotting with the indicated Abs. Data are representative of three replicate experiments.

Article Snippet: The mouse lung epithelial (MLE12) cell line and Jurkat T cell line were purchased from American Type Culture Collection (Manassas, VA).

Techniques: In Vitro, Ubiquitin Proteomics, Incubation, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, SDS Page

Journal: JCI Insight

Article Title: Sine oculis homeobox homolog 1 plays a critical role in pulmonary fibrosis

doi: 10.1172/jci.insight.142984

Figure Lengend Snippet: ( A ) Western blot showing SIX1 protein overexpression using pCDNA-m Six1 vector ( Six1 OE) in MLE-12 cells compared with GFP-encoding pCDNA control vector-transfected MLE12 cells. ( B ) RNA-Seq data normalized count numbers ± SD comparing GFP control MLE12 cells ( n = 3) to Six1 OE ( n = 3); *** P ≤ 0.001 and **** P ≤ 0.0001 refer to 2-way ANOVA comparisons between pcDNA and Six1 pcDNA with the 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post hoc test. ( C ) Representation depicting the Six1 binding sequence (TCAGG) in the mouse and human Mif promoters. ( D and E ) qPCR showing increased expression of Six1 ( D ) and Mif ( E ) in Six1 OE cells. ( F ) Western blots showing increase in Six1 and MIF protein levels in Six1 OE cells compared with GFP controls. ( G and H ) Ratios of Gaussian luciferase/secreted embryonic alkaline phosphatase (G-Luc/SEAP) showing levels of Mif -promoter activity from Six1 OE cells compared with GFP controls at 12 hours ( G ) or 24 hours ( H ) of stimulation. ** P ≤ 0.01 by Student’s t tests with Welch correction.

Article Snippet: Mouse alveolar lung epithelial cell line MLE12 (ATCC-CRL-2110, ATCC; ref. ) was used to assess Six1 function in vitro.

Techniques: Western Blot, Over Expression, Plasmid Preparation, Control, Transfection, RNA Sequencing, Binding Assay, Sequencing, Expressing, Luciferase, Activity Assay