Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: USP20 directly interacts with GPX4. ( A ) Immunoblot analysis of target proteins in sorafenib-resistant 769-P and A549 cells following 24 h treatment with specific deubiquitinase inhibitors. ML-323: 10 μM, GSK2643943A: 10 μM, XL177A: 10 μM, Spautin-1: 10 μM, IU1: 10 μM, AZ1: 10 μM, MF-094: 10 μM, LDN-57444: 10 μM, TCID: 10 μM, PR-619: 10 μM, BAY11-7082: 10 μM, EOAI3402143: 2.5 μM, ML364: 10 μM, b-AP15: 0.5 μM. ( B ) HEK293T cells were transfected with GST-GPX4 and Flag-USP. The cell lysate was applied with GST pull-down, then analyzed with immunoblot for indicated proteins. ( C ) 769-P and A549 cells were subjected to immunoprecipitate with anti-GPX4 antibodies, then analyzed with immunoblot for indicated proteins. ( D ) Overview of USP20 structures. ( E-F ) HEK293T cells transfected with the indicated USP20 structures and Flag-GPX4 were subjected to pull-down with GSH beads or immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( G ) 769-P and A549 cells were subjected to subcellular fractionation, then analyzed with immunoblot for indicated proteins. ( H ) Overview of GPX4 isoforms structures. ( I ) HEK293T cells transfected with the indicated GPX4 isoforms and GST-USP20 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( J ) HEK293T cells transfected with the GST-USP20 WT/C154S and Flag-GPX4 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins.

Article Snippet: Primary antibodies against USP20 (cat. no. 17491-1-AP; in ), HA-Tag (cat. no. 51064-2-AP), FLAG-Tag (cat. no. 20543-1-AP), GST-Tag (cat. no. 66001-2-Ig), β-Actin (cat. no. 66009-1-Ig), His-Tag (cat. no. 66005-1-Ig), GPX4(cat.no.67763-1-Ig), ACSL4 (cat. no. 22401-1-AP), SLC7A11 (cat. no. 26864-1-AP), FSP1 (cat. no. 20886-1-AP), FTH (cat. no. 11682-1-AP), Vinculin (cat. no. 26520-1-AP), MEK (cat. no. 11049-1-AP), TOMM20 (cat. no. 11802-1-AP), and H3 (cat. no. 17168-1-AP) were purchased from ProteinTech.

Techniques: Western Blot, Transfection, Fractionation

Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: USP20 removes GPX4 K48-linked poly-ubiquitination. ( A ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, GST-USP20 and His-Ub. ( B ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, GST-USP20 WT/C154S and His-Ub. ( C ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, and His-Ub under the condition of the indicated concentration of GSK2643943A. ( D ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in USP20 knockdown HEK293T cells transfected with Flag-GPX4 and His-Ub. ( E ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, HA-USP20 and His-Ub WT or His-Ub K48−only . ( F ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, HA-USP20 and His-Ub WT or His-Ub K48R .

Article Snippet: Primary antibodies against USP20 (cat. no. 17491-1-AP; in ), HA-Tag (cat. no. 51064-2-AP), FLAG-Tag (cat. no. 20543-1-AP), GST-Tag (cat. no. 66001-2-Ig), β-Actin (cat. no. 66009-1-Ig), His-Tag (cat. no. 66005-1-Ig), GPX4(cat.no.67763-1-Ig), ACSL4 (cat. no. 22401-1-AP), SLC7A11 (cat. no. 26864-1-AP), FSP1 (cat. no. 20886-1-AP), FTH (cat. no. 11682-1-AP), Vinculin (cat. no. 26520-1-AP), MEK (cat. no. 11049-1-AP), TOMM20 (cat. no. 11802-1-AP), and H3 (cat. no. 17168-1-AP) were purchased from ProteinTech.

Techniques: Ubiquitin Proteomics, Transfection, Concentration Assay, Knockdown

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Transgelin expression pattern in colorectal cancer. (A) Transgelin expression in normal colonic tissues. (B) Transgelin expression in colorectal cancer tissues. (C) Transgelin expression in cancer tissues. (D) Fluorescent immunostaining showed that transgelin was expressed in desmin‐positive smooth muscles as well as in other cancer stromal regions; transgelin, desmin are shown in dark green, green, and purple, respectively. (E) Immunostaining results for transgelin (dark green), smooth muscle Actin (SMA; red), and 4′,6‐diamidino‐2‐phenylindole (DAPI; purple) in the cancer cell stroma. (F) Immunostaining for AE1/3 (green), transgelin (dark green), and DAPI (purple).

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Expressing, Immunostaining, Muscles

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Knockdown of transgelin in cSPFs does not alter the expression of α‐SMA, COL1A1, and TNC. (A) Comparison of transgelin and α‐SMA expression levels in cSPFs under unstimulated (blue) and stimulated (orange) conditions. Data are shown as mean ± standard deviation ( n = 3). ** p < 0.01. B Unstimulated and stimulated conditions, with sh‐Luc, sh‐transgelin #1 (#1), or sh‐transgelin #2 (#2), and transgelin expression was quantified using reverse transcription polymerase chain reaction. Data are shown as mean ± standard deviation ( n = 3). ** p < 0.01. (C) Protein levels of transgelin were compared using western blotting following transfection with sh‐Luc, sh‐transgelin #1, or sh‐transgelin #2 in cSPF‐stimulated and unstimulated conditions. (D) Changes in α‐SMA, COL1A1, and TNC expression in cSPFs transfected with sh‐Luc, sh‐transgelin #1, or sh‐transgelin #2 under unstimulated conditions with the cancer cell‐culture supernatant (blue) and after stimulation with cancer cell‐culture supernatants (orange). Different treatment groups were compared using a two‐tailed t ‐test. ** p < 0.01. cSPF: Colonic subperitoneal fibroblast; α‐SMA: Alpha‐smooth muscle Actin; COL1A1: Collagen type I alpha 1; TNC: Tenascin C; sh‐Luc: Short hairpin RNA targeting luciferase.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Knockdown, Expressing, Comparison, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Transfection, Cell Culture, Two Tailed Test, shRNA, Luciferase

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Transgelin knockdown suppresses cSPF‐induced contractility in vitro and tumor growth by co‐transplantation of cSPFs and DLD‐1 cells in vivo. (A) To establish the conditions for the contraction assay, five cell counts were performed: 5 × 10 4 , 1 × 10 5 , 2 × 10 5 , 4 × 10 5 , and 8 × 10 5 . No stimulation by the cancer cell‐culture supernatant (blue) and stimulation by the cancer cell‐culture supernatant (orange). Data are shown as mean ± standard deviation ( n = 3) of at least three independent experiments. (B) The results of the contraction assay under unstimulated conditions with cancer cell‐culture supernatant with sh‐Luc (Luc), sh‐transgelin #1 (#1), or sh‐transgelin #2 (#2). Data are shown as mean ± standard deviation ( n = 5) of at least two independent experiments. * p < 0.05. (C) The results of the contraction assay under stimulation with the cancer cell‐culture supernatant are shown. Data are shown as mean ± standard deviation ( n = 5) of at least two independent experiments. * p < 0.05. (D) Schematic illustration of the experiment. The left, middle, and right panels show the groups implanted with DLD‐1 cells only, co‐implanted with DLD‐1 cells and cSPFs, and co‐implanted with DLD‐1 cells and cSPFs with transgelin knockdown, respectively. (E) The growth of tumors was compared with the co‐transplantation of DLD‐1 cells, cSPFs (orange), compared with the transplantation of DLD‐1 cells only (blue), and co‐transplantation (gray) of DLD‐1 cells with cSPFs with knockdown. Data are presented as mean (tumor volume) ± standard deviation, n = 9 per group. (F) In sh‐transgelin #2, co‐transplantation (gray) of transgelin‐knockdown cSPFs and DLD‐1 cells suppressed the tumor growth‐promoting effect. Data are presented as mean (tumor volume) ± standard deviation, n = 12 per group. (G) Photograph of mice 4 weeks following transplantation. cSPF, colonic subperitoneal fibroblast; sh‐Luc, short hairpin RNA targeting luciferase.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Knockdown, In Vitro, Transplantation Assay, In Vivo, Contraction Assay, Cell Culture, Standard Deviation, shRNA, Luciferase

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: RNA sequencing results in cSPFs with knockdown of transgelin before and after CM stimulation. (A) Experimental flow diagram. The stimulated and unstimulated cSPFs of the control and transgelin‐knockdown groups were assessed ( n = 3 each; total n = 12). (B) Principal component analysis of the correlation between expression levels showed a correlation with inter‐individual effects rather than knockdown. (C) Heatmap analysis showed a correlation with inter‐individual effects rather than with transgelin‐knockdown effects. cSPF, colonic subperitoneal fibroblast; CM, conditioned medium.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: RNA Sequencing, Knockdown, Control, Expressing

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Assessment of transgelin expression and progression‐free survival in patients with colorectal cancer. (A) A total of 359 of 388 colorectal cancer surgery cases were analyzed after excluding those that met the exclusion criteria. (B) The assessment of the transgelin‐positive area ratio and the α‐SMA‐positive area ratio in the tissue core showed a positive correlation. (C) A high transgelin‐positive area ratio is significantly associated with poor prognosis. (D) A high α‐SMA‐positive area ratio is significantly associated with poor prognosis. α‐SMA, alpha‐smooth muscle Actin; PFS, progression‐free survival.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Expressing