l132 cells  (ATCC)

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1 interacts with ZEB1, increasing its protein level Western blots showing the EMT-TFs in cells transfected with shCTRL or sh GTSE1 with and without (A) IR or (B) TGF-β treatment. Small values above each blot reflect the relative intensity of the target proteins normalized to β-actin. (C) Heatmaps show the protein levels of EMT-TFs in IR-treated L132 and TGF-β-treated HPF cells as a fold change from the naive cell control (left). The IP heatmap (right) shows the GTSE1-binding ratio of EMT-TF proteins as a fold change to the total protein levels. (D) Immunoblots showing the interaction between Flag-tagged GTSE1 and ZEB1 in HEK 293T cells (left) and the interaction between endogenous GTSE1and ZEB1 in L132 cells (right). (E) Immunofluorescence staining for GTSE1 and ZEB1 in L132 cells 24 h after TGF- β treatment. (F) PLA confirms endogenous GTSE1–ZEB1 interactions in cells transfected with shCTRL or sh GTSE1 and treated with TGF-β. Interactions with the target proteins are indicated as red dots. Cell nuclei were counterstained with DAPI. Representative fluorescence images (G) from BLM- or IR-induced PF mouse tissues and (H) from IPF patient tissues. (I) Correlation between GTSE1 and ZEB1 expression in IPF patients (Normal: n = 24, IPF: n = 24). All graphs indicate the mean ± SEM. The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: To determine the EMT or FMT phenotype, we used both epithelial and fibroblast cells as previously described., , , L132 human pulmonary epithelial cells and WI-38 human fetal lung fibroblast cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Transfection, Control, Binding Assay, Immunofluorescence, Staining, Fluorescence, Expressing

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: Increase protein stability of ZEB1 mediates GTSE1-induced EMT (A) RT-PCR in cells transfected with shCTRL or sh GTSE1 with and without 8 Gy of IR. Small values above each blot reflect the relative intensity of the target mRNAs normalized to the endogenous control, gapdh . (B) Measurement of ZEB1 protein stability under a protein synthesis blockade from CHX. L132 cells were transfected with sh GTSE1 or Flag-GTSE1 and treated with 100 μg/mL CHX for various time periods. The graph shows the relative levels of ZEB1 protein as a fold change from its initial level. (C and D) The extent of ubiquitination of the ZEB1 protein was confirmed by immunoprecipitation and immunoblot analyses. (C) HEK 293T cells transfected with Flag-GTSE1 at 24 h. Cells were treated with 10 μM MG132 for 8 h. (D) siGTSE1 was co-transfected with HA-tagged ubiquitin in HEK 293T cells. (E) CDH1 promoter activity in HEK 293T cells was measured using a luciferase reporter assay after siGTSE1 transfection for 24 h. (F) ChIP assay was performed in GTSE1-deficient L132 cells. The chromatin was immunoprecipitated with an anti-ZEB1 antibody. Each precipitated DNA was analyzed by quantitative PCR using promoter-specific primers for CDH1 . (G) Immunoblots for EMT markers and (H) IF staining for F-actin in L132 cells co-transfected with Flag-tagged GTSE1 and si ZEB1 . (I) Cell migration of GTSE1-deficient L132 cells was assessed using a wound-healing assay after 24 and 48 h, with and without transfection with Flag-tagged ZEB1. Results are presented as a percentage of wound closure compared with the original volume. All graphs indicate the mean ± SEM. The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: To determine the EMT or FMT phenotype, we used both epithelial and fibroblast cells as previously described., , , L132 human pulmonary epithelial cells and WI-38 human fetal lung fibroblast cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Activity Assay, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Staining, Migration, Wound Healing Assay

Journal: Molecular Therapy

Article Title: GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition

doi: 10.1016/j.ymthe.2024.09.029

Figure Lengend Snippet: GTSE1 binds to the non-phosphorylated form of ZEB1 and blocks its degradation (A) A diagram illustrating the ZEB1 mutants. The phospho-deficient mutant (S585A) replaces serine 585 with alanine, and the phosphor-mimetic mutant (S585E) replaces serine 585 with aspartic acid. (B) V5-tagged GTSE1 was co-transfected with the Flag-tagged ZEB1 mutant forms in HEK 293T and (C) L132 cells. Phosphorylation of each ZEB1 mutant was analyzed by immunoprecipitation with an anti-V5 agarose affinity gel antibody and detected with a western blot analysis. PLA confirmed the interaction between the V5-tagged GTSE1 and Flag-tagged ZEB1 mutant forms. Interactions with the target proteins are indicated as red dots. Cell nuclei were counterstained with DAPI (blue). (D) Ubiquitination of Flag-tagged ZEB1 was analyzed by immunoprecipitation with an anti-HA agarose affinity gel antibody and detected with a western blot analysis. V5-tagged GTSE1 and the Flag-tagged ZEB1 mutant forms were co-transfected with HA-tagged ubiquitin into HEK 293T cells for 24 h. (E) HEK 293T cells were transfected with Flag-tagged ZEB1 mutant forms and siCTRL or si GTSE1 and treated with 100 μg/mL CHX for various time periods. The graph shows the relative protein levels as a fold change from the initial Flag-tagged ZEB1 protein level. (F) L132 cells were transfected with siCTRL, si ATM , or si GTSE1 and exposed to 10 Gy of IR. Protein expression was then analyzed by western blotting. (G) The interaction between Flag-tagged ZEB1 and ATM or GTSE1 was confirmed by immunoprecipitation and immunoblot analyses. Flag-tagged ZEB1 and siATM or siGTSE1 were co-transfected with HEK 293T cells for 24 h. (H) L132 cells were transfected with siCTRL, siATM, or siGTSE1 and treated with 100 μg/mL of CHX for various time periods. The graph shows the relative levels of ZEB1 protein. (I) Immunoblots indicate protein level variations in response to IR. All graphs indicate the mean ± SEM ( n = 3/group). The fold change was calculated relative to the control. ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicates p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: To determine the EMT or FMT phenotype, we used both epithelial and fibroblast cells as previously described., , , L132 human pulmonary epithelial cells and WI-38 human fetal lung fibroblast cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Mutagenesis, Transfection, Phospho-proteomics, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing, Control

Journal: Briefings in Bioinformatics

Article Title: Standigm ASK™: knowledge graph and artificial intelligence platform applied to target discovery in idiopathic pulmonary fibrosis

doi: 10.1093/bib/bbae035

Figure Lengend Snippet: Phenotypes of designated siRNA-transfected cells. ( A ) Effects of siRNAs on F-actin organization in L132 cells in the presence of TGFβ for 24 h. F-actin was visualized by Alexa Fluor™ 488 Phalloidin. The magnification is 40′. ( B ) The migration capacity of siRNA-transfected cells with or without treatment with TGFβ was detected using a wound-healing assay. ( C ) Cell proliferation upon transfection of siRNAs with or without TGFβ treatment was determined by MTT assay ( * P < 0.05, * * P < 0.01, * * * P < 0.001 compared with control cells; # P < 0.05, # # P < 0.01, # # # P < 0.001 compared with cells treated with TGFβ alone).

Article Snippet: A human normal lung epithelial cell line (L132) and HEK 293T cells were supplied by the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI (Gibco, Gaithersburg, MD, USA) or DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified 5% CO 2 incubator.

Techniques: Transfection, Migration, Wound Healing Assay, MTT Assay, Control