human cell lines mrc5  (ATCC)

Journal: iScience

Article Title: TRIM36 inhibits lung fibroblast activation and pulmonary fibrosis through the degradation of phospho-AKT1

doi: 10.1016/j.isci.2025.113775

Figure Lengend Snippet: TRIM36 inhibits lung fibroblast proliferation, migration, and differentiation (A) The efficiency of TRIM36 overexpression was detected by immunoblotting. (B and C) Cell proliferation assessed by CCK-8 assay in MRC5 and IMR-90 cells transfected with indicated plasmids ( n = 3). (D and E) Cell proliferation evaluated by EdU assay in MRC5 and IMR-90 cells transfected with indicated plasmids ( n = 3). Positive cell numbers are quantified in bar graphs. Scale bars: 100 μm. (F and G) Cell migration evaluated by Transwell assay in MRC5 and IMR-90 cells transfected with indicated plasmids ( n = 3). Migrated cell numbers are quantified in bar graphs. Scale bars: 100 μm. (H and I) MRC5 and IMR-90 cells were transfected with the indicated plasmids and treated with dimethyl sulfoxide (DMSO) or TGF-β1 (10 ng/mL) for 24 h before lysis. Immunoblot analysis was performed using indicated antibodies. (J and K) The expression intensity of Fibronectin 1, Collagen 1, and α-SMA in (H) and (I) were quantified by densitometry, with GAPDH as a normalizer. Each data point represents the test result of one sample. The lanes in western blots correspond to technical replicates, and similar results were repeated in three biologically independent experiments. Data are shown as the means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human cell line: MRC5 , ATCC , Cat# CCL-171.

Techniques: Migration, Over Expression, Western Blot, CCK-8 Assay, Transfection, EdU Assay, Transwell Assay, Lysis, Expressing

Journal: iScience

Article Title: TRIM36 inhibits lung fibroblast activation and pulmonary fibrosis through the degradation of phospho-AKT1

doi: 10.1016/j.isci.2025.113775

Figure Lengend Snippet: TRIM36 destabilizes AKT1 (A) Endogenous interaction between TRIM36 and AKT1. MRC5 and IMR-90 cells were collected and lysed. The extracts were IP using indicated antibodies. (B and C) Schematic diagram indicated the structure of TRIM36 (B) and AKT1 (C) and their deletion mutants. FLAG-TRIM36 WT or deletion mutants were co-transfected with HA-AKT1 in human embryonic kidney 293T (HEK293T) cells (B). FLAG-AKT1 WT or deletion mutants were co-transfected with HA-TRIM36 in HEK293T cells (C). HEK293T cells were collected and lysed. IP was performed using anti-FLAG magnetic beads and analyzed by immunoblotting. (D) TRIM36 shorten AKT1 protein half-life. Cells ectopically expressing TRIM36 were treated with CHX (100 μg/mL) for indicated times. The cells were collected and analyzed by immunoblotting with the indicated antibodies. (E) The expression intensities of AKT1, p-AKT1(T308), and p-AKT1(S473) at each time point in (D) were quantified by densitometry, with GAPDH as a normalizer. (F) TRIM36 overexpression cells were treated with TGF-β1 (10 ng/mL). The cells were collected and analyzed by immunoblotting with the indicated antibodies. Similar results were repeated in three biologically independent experiments. Data are shown as the means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human cell line: MRC5 , ATCC , Cat# CCL-171.

Techniques: Transfection, Magnetic Beads, Western Blot, Expressing, Over Expression

Journal: iScience

Article Title: TRIM36 inhibits lung fibroblast activation and pulmonary fibrosis through the degradation of phospho-AKT1

doi: 10.1016/j.isci.2025.113775

Figure Lengend Snippet: TRIM36 promotes proteasomal degradation of AKT1 via K48-linked ubiquitination at lysine 268 (A) MRC5 and IMR-90 cells ectopically expressing TRIM36 were treated with MG132 or DMSO for 5 h. Cells were collected and analyzed by immunoblotting with the indicated antibodies. (B and C) HEK293T cells were transfected with the indicated plasmids and treated with MG132 for 5 h. The cells were collected and lysed in RIPA buffer. Immunoprecipitated AKT1 was analyzed by immunoblotting with the indicated antibodies. (D) The polyubiquitination of AKT1 in the presence of TRIM36 and either WT ubiquitin or its mutants. HEK293T cells were transfected with the indicated plasmids. 48 h after transfection, cells were treated with MG132 for 5 h before lysis. IP and immunoblot analysis were performed as described in (B). (E) The K268 sites of AKT1 are highly conserved across species. (F) The polyubiquitination of AKT1 WT and its mutants. HEK293T cells were transfected with the indicated plasmids. IP and immunoblot analysis were performed as described in (B). Similar results were repeated in three biologically independent experiments.

Article Snippet: Human cell line: MRC5 , ATCC , Cat# CCL-171.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Transfection, Immunoprecipitation, Lysis

Journal: iScience

Article Title: TRIM36 inhibits lung fibroblast activation and pulmonary fibrosis through the degradation of phospho-AKT1

doi: 10.1016/j.isci.2025.113775

Figure Lengend Snippet: TRIM36 preferentially ubiquitinates the phospho-AKT1 (T308/S473) (A) HEK293T cells were transfected with FLAG-TRIM36 and HA-AKT1 plasmids for 48 h. The cells lysates were treated with λ protein phosphatase for 30 min, followed by IP using anti-FLAG magnetic beads and immunoblot analysis. (B and C) HEK293T cells were transfected with the indicated plasmids and treated with 2 μM MK2206 or 4 μg/mL SC79. IP and immunoblot analysis were performed as described in (A). (D) HEK293T cells were transfected with vectors expressing HA-AKT1 WT or its mutants and progressive increasing doses of Myc-TRIM36. Cells were collected and analyzed by immunoblotting with the indicated antibodies. (E) The polyubiquitination of AKT1 WT or its mutants. HEK293T cells were transfected with indicated plasmids and treated with MG132 for 5 h before lysis. IP and immunoblot analysis were performed as described in ( B). (F) Interaction between TRIM36 and AKT1 WT or its mutants. HEK293T cells were transfected with indicated plasmids. IP and immunoblot analysis were performed as described in (A). (G) Glutathione S-transferase (GST) pull-down assays indicated the interaction between GST-TRIM36 and His-AKT1 WT or its mutants. (H) Endogenous interaction between TRIM36 and phospho-AKT1 (S473). MRC5 and IMR-90 cells were collected and lysed. The extracts were IP using phospho-AKT1 (S473) antibody. Immunoblot analysis was performed as described in (A). Similar results were repeated in three biologically independent experiments.

Article Snippet: Human cell line: MRC5 , ATCC , Cat# CCL-171.

Techniques: Transfection, Magnetic Beads, Western Blot, Expressing, Lysis

Journal: iScience

Article Title: TRIM36 inhibits lung fibroblast activation and pulmonary fibrosis through the degradation of phospho-AKT1

doi: 10.1016/j.isci.2025.113775

Figure Lengend Snippet: TRIM36 inhibits lung fibroblast activation by suppressing AKT1 activity (A) MRC5 cells were transfected with the indicated plasmids and treated with DMSO or MK2206 for 24 h before lysis. Immunoblot analysis was performed using indicated antibodies. (B–E) Edu and Transwell assays were performed in the cells from (A). Positive cell numbers of (B) are quantified in bar graphs. The cell number of (D) was counted and exhibited in the bar graphs. n = 3. Scale bars: 100 μm. (F) MRC5 cells were transfected with TRIM36 and AKT WT or its mutants. Immunoblot analysis was performed using the indicated antibodies. (G–J) Edu and Transwell assays were performed in the cells from (F). Positive cell numbers of (G) are quantified in bar graphs. The cell number of (I) was counted and exhibited in the bar graphs. n = 3. Scale bars: 100 μm. Each data point represents the test result of one sample. (A and F) Three biological replicates (BioRep). Data are shown as the means ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human cell line: MRC5 , ATCC , Cat# CCL-171.

Techniques: Activation Assay, Activity Assay, Transfection, Lysis, Western Blot