mrc5 human lung normal cells  (ATCC)

Journal: Translational Cancer Research

Article Title: Metaxins regulate cancer stem cell-like properties in H1299 cells

doi: 10.21037/tcr-2025-aw-2341

Figure Lengend Snippet: Methylation of the MTX1 promoter region in MRC5 and H1299 cells. (A) RT-PCR and western blot analysis of the expression level of MTX1 and MTX2 in MRC5 and H1299 cell lines. (B) Sequence of MTX1 and bisulfite-converted sequences. Each Y shown in the red character indicates the methylated positions in cells. (C) Diagram of pyrosequencing in MRC5 and H1299 cell lines. Each colored box indicates the position of the three Y bases, and the corresponding methylation percentage is shown in the panel. The figure compared the fractions at the methylated CpG positions. The x-axis represents the nucleotides dispensation sequence, and the y-axis indicates the detected signal intensity. Data represent mean ± SD using two-way ANOVA, followed by Tukey’s multiple comparisons test to determine pairwise significance. ns, not significant; **, P<0.01; ***, P<0.001, MRC5 vs. H1299 group. ANOVA, analysis of variance; CpG, cytosine-phosphate-guanine; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation; W.B, western blotting.

Article Snippet: Human lung fibroblast MRC5 (RRID:CV0440) and cancer H1299 (RRID:CV0060) cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Methylation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Sequencing, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: DDI2 depletion leads to autophagy induction (A) Western blot analysis confirms the generation of NIH-3T3 DDI2 KO cells. (B) TEM images of NIH-3T3 control and DDI2 KO cells. N: nucleus. Yellow arrows: early autolysosomes. Blue arrows: late autolysosomes. Scale bars, 2 μm. (C) Autophagy flux analysis in NIH-3T3, MRC5, HAP1, ES1, EW16, and MIA PaCa-2 cells, either control or DDI2-deficient. Cells are treated with CQ (60 μM) and evaluated for LC3B-II protein levels through immunoblotting. β-Actin or GAPDH is used as the loading control. Quantification of LC3B-II protein levels is normalized to the respective loading controls, and corresponding densitometric bar graphs are shown. The molecular weights of the proteins analyzed are as follows: DDI2 (∼45 kDa), LC3B-II (∼16 kDa), β-actin (∼45 kDa), and GAPDH (∼36 kDa). Three biological replicates for each cell line are used to perform Western blotting. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Western Blot, Control, Molecular Weight, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: DDI2 deficiency increases CCN1 protein levels without affecting CCN1 transcription (A) Volcano plots show protein abundances in HAP1 cells in the absence of DDI2. p -values are calculated using a two-tailed unpaired t test with unequal variance. The negative log10 of the p -values is plotted on the Y axis, and the log2 fold changes are plotted on the X axis. The plots are generated using VolcaNoseR. (B) Western blot analysis of CCN1 protein levels in NIH-3T3, MRC5, ES1, EW16 and MIA PaCa-2 control, or DDI2-deficient cells treated with or without 60 μM CQ for 24 h. β-Actin or GAPDH is used as a loading control. Quantification of CCN1 protein levels is normalized to the respective loading controls, and corresponding densitometric bar graphs are shown. The molecular weight of CCN1 is 41 kDa. (C) CCN1 expression in MRC5 control and DDI2 KO cells is assessed by immunofluorescence staining. Confocal microscopy is applied to visualize CCN1 localization (green), and CCN1 fluorescence intensity is quantified using ImageJ software. Nuclei are shown in blue through staining with DAPI. Scale bars, 10 μm. (D) qRT-PCR analysis is performed to assess CCN1 mRNA levels in NIH-3T3, MRC5, and MIA PaCa-2 DDI2 KO and control cells, following treatment with either vehicle or 60 μM CQ for 24 h. Gene-specific primers are used as described in the , with 18S rRNA or GAPDH for normalization. Three biological replicates for each cell line are used to perform qRT-PCR and Western blotting. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Two Tailed Test, Generated, Western Blot, Control, Molecular Weight, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Software, Quantitative RT-PCR, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: CCN1 is required and sufficient to induce autophagy (A) Autophagy flux analysis following CCN1 knockdown via siRNA in NIH-3T3 DDI2 KO cells and CCN1 knockout using viral particles in MIA PaCa-2 DDI2 KO cells, with the subsequent treatment of the cells with CQ (60 μM) for 24 h. For siRNA transfection, GAPDH is used as a positive control. (B) NIH-3T3, MRC5, and MIA PaCa-2 cells, either control or overexpressing CCN1, are treated with CQ (60 μM) for 24 h, and autophagy flux is measured by Western blot. (C) Autophagy flux analysis in MIA PaCa-2 DDI2 KO cells following CCN1 overexpression, treated with or without CQ (60 μM) for 24 h. (D) MIA PaCa-2 controls, DDI2 KO , and DDI2 KO cells overexpressing CCN1 are treated with or without 50 nM CFZ for 16 h, and cell lysates are analyzed by Western blot using the indicated antibodies. (E) MIA PaCa-2 controls, DDI2 KO , and DDI2 KO cells overexpressing CCN1 are assessed for cell viability using the luminescent CellTiter-Glo assay. β-actin or GAPDH is used as a loading control for Western blotting, and corresponding densitometric bar graphs are shown. Quantification of LC3B-II protein levels is normalized to the respective loading controls. Each experiment is performed in three biological replicates for Western blotting and six replicates for the cell viability assay. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Tukey’s or Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, not significant. Corresponding immunoblots including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Knockdown, Knock-Out, Transfection, Positive Control, Control, Western Blot, Over Expression, Glo Assay, Viability Assay, Molecular Weight, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: Intracellular accumulation of CCN1 induces autophagy through ROS production (A) MRC5 WT cells are treated with either 15 μg/mL BFA (bottom) or left untreated (top), followed by immunofluorescent staining for CCN1. The stained samples are imaged using confocal microscopy, with CCN1 fluorescence shown in green and nuclei labeled in blue using DAPI. Images are analyzed using ImageJ software. Scale bars, 10 μm. (B) Western blot analysis of intracellular CCN1 and LC3B-II protein levels in DDI2 KO and control cells treated with 15 μg/mL BFA, 60 μM CQ, or a combination of both for 24 h. (C) Measurement of autophagic flux in CCN1 KO and control cells following treatment with 15 μg/mL BFA, 60 μM CQ, or both for 24 h. (D) Western blot analysis of conditioned media to evaluate secreted CCN1, and validate the generation of MRC5 CCN1 ΔSP and WT CCN1-overexpressing cells. β-Actin from corresponding cell lysates is used as a loading control, reflecting equal numbers of plated cells. (E) Measurement of autophagic flux in MRC5 CCN1 ΔSP and WT CCN1 cells compared to control cells following treatment with 60 μM CQ for 24 h. (F) Western blot analysis of LC3B-II protein levels in CCN1 OE and control cells treated with 15 μg/mL BFA, 0.5 mM NAC, or a combination of both for 24 h. β-Actin or GAPDH is used as a loading control for all Western blots, and corresponding densitometric bar graphs are shown. Quantification of LC3B-II and CCN1 protein levels is normalized to the respective loading controls. Three biological replicates for each cell line are used to perform Western blotting. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Staining, Confocal Microscopy, Fluorescence, Labeling, Software, Western Blot, Control, Molecular Weight, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: DDI2 interacts with CCN1 and p97 (A) MRC5 and MIA PaCa-2 cells, either DDI2 KO or control, are treated with DMSO or CB-5083 (10 μM for 3 h). CCN1 protein levels are analyzed by immunoblotting using antibodies specific to DDI2, CCN1, and β-Actin, and representative blots with corresponding densitometric bar graphs are shown. (B) HEK293 cells transiently expressing protease-dead DDI2 (Flag-dRVP DDI2) are subjected to immunoprecipitation using anti-FLAG beads, followed by immunoblotting with antibodies specific to DDI2, CCN1, p97, and RAD23A. Lysate lanes are loaded with 5% of the total input used for immunoprecipitation. The molecular weights of the proteins analyzed are as follows: P97/VCP (89 kDa), RAD23A (52 kDa). (C) Representative confocal immunofluorescence microscopy images show the co-localization of DDI2 (red) and CCN1 (green) in wild-type MRC5 and MIA PaCa-2 cells. The nuclei are visualized by staining with DAPI (blue). Scale bars, 10 μm. Three biological replicates for each cell line are used to perform Western blotting. Statistical significance of each condition compared to the indicated control or treatment is determined using unpaired Student’s t test or two-way ANOVA with Šídák’s post hoc test, as appropriate. Data are represented as mean ± SEM. Significance levels are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Control, Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Microscopy, Staining, Molecular Weight, Marker

Journal: iScience

Article Title: Loss of DDI2 rewires proteostasis through CCN1-driven compensatory autophagy

doi: 10.1016/j.isci.2026.115056

Figure Lengend Snippet: CCN1 associates with the lysosome, and DDI2 directs CCN1 toward lysosomal degradation (A) Co-immunoprecipitation of CCN1 and LAMP1 is performed in HEK293 cells without or (B) with CCN1 overexpression, using protein A/G agarose beads. Western blot analysis is applied with specific antibodies for CCN1 and LAMP1 under high- and low-contrast imaging conditions. The molecular weight of LAMP1 is 90–120 kDa (glycosylated). (C) Immunofluorescence and confocal microscopy are used to assess CCN1 colocalization with LAMP1 in MIA PaCa-2 and MRC5 wild-type cells. CCN1 is stained green in MIA PaCa-2 cells and red in MRC5 cells, while LAMP1 is stained red in MIA PaCa-2 cells and green in MRC5 cells, with nuclei visualized by DAPI staining (blue). Scale bars, 10 μm. (D) Validation of CCN1-V5 tag overexpression in MRC5 DDI2 KO cells compared to the control by Western blotting. (E) Confocal immunofluorescence microscopy images of MRC5 DDI2 KO cells overexpressing CCN1-V5 tag, and MRC5 WT cells overexpressing CCN1-V5 tag. Fixed cells are probed for CCN1-V5 tag (red) and LAMP1 (green) and stained with DAPI (blue) to visualize nuclei. Scale bars, 10 μm. Corresponding immunoblots, including molecular weight marker lanes, are provided in .

Article Snippet: Human: MRC5 , ATCC , CCL-171.

Techniques: Immunoprecipitation, Over Expression, Western Blot, Imaging, Molecular Weight, Immunofluorescence, Confocal Microscopy, Staining, Biomarker Discovery, Control, Microscopy, Marker

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: ANGPTL4 is upregulated in senescent cells. (A) Venn diagram showing genes commonly upregulated between 6 different senescence transcriptome datasets: RAS‐induced senescence in IMR90 fibroblasts (IMR_RAS); cigarette smoke condensate‐induced senescence in MRC5 fibroblasts (MRC5_CSC); etoposide‐induced senescence in WI38 fibroblasts (WI38_ET); human umbilical vein endothelial cells in replicative senescence (HUVEC_RS); MEK‐induced senescence in human mammary epithelial cells (hMEC_MEK); and H 2 O 2 ‐induced senescence in astrocytes (Astrocytes_H 2 O 2 ). ANGPTL4, the only gene upregulated in all conditions is indicated. (B) Relative mRNA expression of P21 , KI67 and ANGPTL4 genes by using RT‐qPCR, in MRC5/RAF:ER cells untreated (CTRL) or treated with 4‐OHT (RAF) for Oncogene Induced Senescence (OIS), in p20‐p23 (EARLY) and p38‐p41 (LATE) MRC5 passages for replicative senescence (RS), and in MRC5 untreated (NT) or treated with bleomycin (BLEO) for therapy‐induced senescence (TIS). Mean ± SEM of n = 3 independent experiments. Paired t‐test results are indicated. (C) Western blot analysis of ANGPTL4 expression in MRC5/RAF:ER not treated (CTRL) or treated with 4‐OHT (OIS). Full‐length ANGPTL4 detected and TUBULIN loading control in whole extract and cleaved ANGPTL4 (c‐ANGPTL4) detected in cell supernatants. Representative image of n = 3 independent experiments.

Article Snippet: MRC5 normal human fibroblasts (ATCC), 293GP (Clonotech) and 293 T (Clontech) were cultured in DMEM (Dubelcco's Modified Eagle's Medium, Life Technologies) supplemented with 10% Fetal Calf Serum (FCS, Life Technologies) and 1% antibiotics (Penicillin, Streptomycin, Gibco).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: ANGPTL4 promotes production of the proinflammatory SASP during senescence. (A) RT‐qPCR analysis of ANGPTL4 and other SASP members without RAF activation (NT) or at different time points after RAF activation, from day 1 (D1) to day 5 (D5), in MRC5/RAF:ER cells. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA tests are shown. (B–D) MRC5/RAF:ER cells were transfected with control (siCTRL) or ANGPTL4 (siANGPTL4) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS). (B) RT‐qPCR analysis of the gene expression of SASP members. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA tests are shown. (C) Western blot analysis of ANGPTL4 and IL6 proteins. Representative image of n = 3 independent experiments. (D, E) Quantification of ANGPTL4 (D) and of IL1A, IL6 and IL8 SASP factors (E) performed by ELISA. Mean protein concentrations measured in cell culture medium are ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are indicated.

Article Snippet: MRC5 normal human fibroblasts (ATCC), 293GP (Clonotech) and 293 T (Clontech) were cultured in DMEM (Dubelcco's Modified Eagle's Medium, Life Technologies) supplemented with 10% Fetal Calf Serum (FCS, Life Technologies) and 1% antibiotics (Penicillin, Streptomycin, Gibco).

Techniques: Quantitative RT-PCR, Activation Assay, Transfection, Control, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: ANGPTL4 acts upstream of IL1A to activate the transcriptional proinflammatory SASP program. (A) MRC5 cells were transfected with control (siCTRL) or siRNA targeting IL1A (siIL1A) during seeding. The days after, cells were infected with control or ANGPTL4 encoding retroviral vectors. Three days later, RNAs were prepared. Relative mRNA expression of ANGPTL4 and proinflammatory SASP member genes was measured by RT‐qPCR. (B) MRC5 cells were transfected with control (siCTRL) or siRNA targeting RELA (siRELA) or ANGPTL4 (siANGPTL4) during seeding. The days after, cells were infected with control or IL1A encoding lentiviral vectors. Three days later, RNAs were prepared. Relative mRNA expression of ANGPTL4, RELA and proinflammatory SASP member genes was measured by RT‐qPCR Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown.

Article Snippet: MRC5 normal human fibroblasts (ATCC), 293GP (Clonotech) and 293 T (Clontech) were cultured in DMEM (Dubelcco's Modified Eagle's Medium, Life Technologies) supplemented with 10% Fetal Calf Serum (FCS, Life Technologies) and 1% antibiotics (Penicillin, Streptomycin, Gibco).

Techniques: Transfection, Control, Infection, Retroviral, Expressing, Quantitative RT-PCR

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: Knockdown of ANGPTL4 inhibits the ability of the SASP to induce activation of human neutrophils, a mark of inflammation. (A, B) Supernatants of MRC5/RAF:ER cells transfected with control (siCTRL), ANGPTL4 (siANGPTL4) or RELA (siRELA) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS) were applied to primary human neutrophils. (A) Analysis of CD63 neutrophils degranulation marker. A representative flow cytometry profile is shown. Histograms on the right panel are the quantification of MFI. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown. (B) Analysis of STAT5 pathway activation according to phosphorylation of STAT5. The left panel displays a representative flow cytometry profile. Histograms on the right panel are the quantification of MFI (Mean Fluorescence Intensity). Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown.

Article Snippet: MRC5 normal human fibroblasts (ATCC), 293GP (Clonotech) and 293 T (Clontech) were cultured in DMEM (Dubelcco's Modified Eagle's Medium, Life Technologies) supplemented with 10% Fetal Calf Serum (FCS, Life Technologies) and 1% antibiotics (Penicillin, Streptomycin, Gibco).

Techniques: Knockdown, Activation Assay, Transfection, Control, Marker, Flow Cytometry, Phospho-proteomics, Fluorescence

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: A hypoxia‐like response mediated by HIF2A upregulates ANGPTL4 during senescence. (A) GSEA plots showing enrichment of the HYPOXIA gene set in 3 senescence models: RAS‐induced senescence in IMR90 (IMR90‐RAS) MEK‐induced senescence in human mammary epithelial cells (hMEC_MEK), and etoposide‐induced senescence in WI38 (WI38‐ETO). Normalized Enrichment Score (NES) and FDR q‐value are indicated. (B, C) MRC5 cells were infected with an empty vector (CTRL), or HIF1A (HIF1A OE) and HIF2A (HIF2A OE) expressing vectors. (B) Relative mRNA expression of HIF1A , HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA values are indicated. (C) Western blot analysis of HIF1A, HIF2A, ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments. (D) HIF2A peaks identified by Cut & Tag at the ANGPTL4 promoter in MRC5 cells infected with HIF2A (HIF2) or control (Babe) retroviral particles. Two independent experiments were performed (HIF2A‐Rep1, HIF2A‐Rep2). Both peaks contain a Hypoxia Response Element (ACGTG). (E) Western blot analysis of ANGPTL4 and the loading control GAPDH during RAF‐induced senescence (OIS). Representative picture of n = 3 independent experiments. (F) MRC5/RAF:ER cells were infected with lentiviral vectors encoding scramble (shSCR) or HIF2A shRNA (shHIF2A) and next treated (+) or not (−) with 4‐OHT to induce senescence (OIS). Relative mRNA expression of HIF2A and ANGPTL4 genes by RT‐qPCR. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown. (G) ChIP‐qPCR assay to assess endogenous HIF2A binding on the ANGPTL4 promoter during OIS induced by RAF. Chromatin fractions derived from 4‐OHT‐treated and untreated MRC5/RAF:ER cells were subjected to immunoprecipitation with anti‐HIF2A antibody or IgG control. Primer sets were designed for regions 2179 bp (distal) and 369 bp (proximal) upstream of the ANGPTL4 TSS, and for Actin promoter as a control. Mean ± SEM of n = 3 independent experiments. Paired t ‐test values are indicated. (H) Western blot analysis of ANGPTL4 and the loading control TUBULIN. Representative picture of n = 3 independent experiments.

Article Snippet: MRC5 normal human fibroblasts (ATCC), 293GP (Clonotech) and 293 T (Clontech) were cultured in DMEM (Dubelcco's Modified Eagle's Medium, Life Technologies) supplemented with 10% Fetal Calf Serum (FCS, Life Technologies) and 1% antibiotics (Penicillin, Streptomycin, Gibco).

Techniques: Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Retroviral, shRNA, ChIP-qPCR, Binding Assay, Derivative Assay, Immunoprecipitation

Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: Cleavage of ANGPTL4 by FURIN is required for its ability to promote the proinflammatory SASP. (A‐C) MRC5/RAF:ER cells were transfected with control (siCTRL) or FURIN (siFURIN) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS). (A) RT‐qPCR analysis against the indicated genes 3 days after 4‐OHT treatment. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown. (B) Western blot analysis of FURIN, ANGPTL4: Full length in whole cell extracts (ANGPTL4) and secreted in the supernatant (cANGPTL4) and IL6. Representative picture of n = 3 independent experiments. (C) RT‐qPCR analysis of the indicated SASP encoding genes. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test values are shown. (D) Relative mRNA expression of FURIN , ANGPTL4 and proinflammatory SASP encoding genes measured by RT‐qPCR in MRC5 cells overexpressing or not ANGPTL4 (ANGPTL4 OE) and transfected with control (siCTRL) or FURIN (siFURIN) siRNA. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown.

Article Snippet: MRC5 normal human fibroblasts (ATCC), 293GP (Clonotech) and 293 T (Clontech) were cultured in DMEM (Dubelcco's Modified Eagle's Medium, Life Technologies) supplemented with 10% Fetal Calf Serum (FCS, Life Technologies) and 1% antibiotics (Penicillin, Streptomycin, Gibco).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, 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 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