Journal: Genes & Diseases

Article Title: Blockade of co-inhibitory receptor immune checkpoint protein TIM3/CD366 augments the anti-cancer activity of CAR-T therapy in solid tumors: An ovarian cancer example

doi: 10.1016/j.gendis.2025.101978

Figure Lengend Snippet: Specific IFN-γ and TNF-α release of T lymphocytes transduced with TIM-3-silenced HER2-specific chimeric antigen receptor (CAR) or HER2-specific CAR. (A, B) TIM-3-silenced CAR-T cells and control T cells were co-incubated with Galectin-9 + or Galectin-9 – SKOV3 tumor cells (E:T ratio 5:1 or 10:1). At 20 h after coculture, a specific enzyme-linked immunosorbent assay was used to analyze the supernatant for IFN-γ cytokine-release. Results were presented as mean ± standard deviation. (C, D) The detection of TNF-α in the same culture supernatant. Results were presented as mean ± standard deviation. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Human cervical cancer cell line HeLa, lentivirus packaging cell line HEK 293TD, and human ovarian cancer cell line SKOV3 were purchased from American Type Culture Collection (Manassas, Virginia, USA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum.

Techniques: Transduction, Control, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Genes & Diseases

Article Title: Blockade of co-inhibitory receptor immune checkpoint protein TIM3/CD366 augments the anti-cancer activity of CAR-T therapy in solid tumors: An ovarian cancer example

doi: 10.1016/j.gendis.2025.101978

Figure Lengend Snippet: TIM-3 silencing augmented the anti-tumor activity of chimeric antigen receptor-T (CAR-T) cells in vivo . 2 × 10 6 SKOV3 tumor cells expressing luciferase were intraperitoneally inoculated in a xenograft mouse model, and 7 days after inoculation, the 2 × 10 6 HER2-specific CAR-T kdTim-3 cells or CAR-T cells, or untreated T cells were intraperitoneally administered. (A, B) Tumor growth was monitored using an in vivo imaging system. (C) Survival curve of 80-day post-treatment. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Human cervical cancer cell line HeLa, lentivirus packaging cell line HEK 293TD, and human ovarian cancer cell line SKOV3 were purchased from American Type Culture Collection (Manassas, Virginia, USA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum.

Techniques: Activity Assay, In Vivo, Expressing, Luciferase, In Vivo Imaging

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: Effects of GEM on hypoxia‐inducible factor 1‐alpha (HIF1A), UBR5, and ovarian cancer Cells. (A) Western blot (WB) analysis of HIF1A and UBR5 expression in SKOV3 cells under hypoxic conditions treated with different concentrations of GEM; (B) WB analysis of HIF1A and UBR5 expression in OVCAR‐3 cells under hypoxic conditions treated with different concentrations of GEM; (C) CCK‐8 assay of cell viability in SKOV3 cells treated with varying concentrations of GEM at 0, 24, and 48 h; (D) CCK‐8 assay of cell viability in OVCAR‐3 cells treated with varying concentrations of GEM at 0, 24, and 48 h; (E) wound healing assay assessing the migration rate of SKOV3 cells treated with different concentrations of GEM at 0 h, 24 h, and 48 h (scale bar: 100 μm); (F) wound healing assay assessing the migration rate of OVCAR‐3 cells treated with different concentrations of GEM at 0, 24, and 48 h (scale bar: 100 μm); (G) transwell invasion assay evaluating the invasive capacity of SKOV3 cells treated with different concentrations of GEM at 0, 24, and 48 h (scale bar: 50 μm); (H) transwell invasion assay evaluating the invasive capacity of OVCAR‐3 cells treated with different concentrations of GEM at 0, 24, and 48 h (scale bar: 50 μm). Data are presented as mean ± SD; all cellular experiments were performed in triplicate. *** p < 0.001, analyzed using ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: Effects of GEM on ovarian cancer proliferation and invasion via HIF1A‐mediated downregulation of UBR5. (A) Western blot (WB) analysis of hypoxia‐inducible factor 1‐alpha (HIF1A) and UBR5 expression levels in SKOV3 cells treated with 20 μM GEM for 48 h; (B) WB analysis of HIF1A and UBR5 expression levels in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (C) CCK8 assay showing cell viability of SKOV3 cells treated with 20 μM GEM for 48 h; (D) CCK8 assay showing cell viability of OVCAR‐3 cells treated with 20 μM GEM for 48 h; (E) wound healing assay assessing the migration rate of SKOV3 cells treated with 20 μM GEM for 48 h (Scale bar: 100 μm); (F) wound healing assay assessing the migration rate of OVCAR‐3 cells treated with 20 μM GEM for 48 h (Scale bar: 100 μm); (G) transwell invasion assay showing the invasive capacity of SKOV3 cells treated with 20 μM GEM for 48 h (Scale bar: 50 μm); (H) transwell invasion assay showing the invasive capacity of OVCAR‐3 cells treated with 20 μM GEM for 48 h (Scale bar: 50 μm); (I) predicted transcription factor binding sites in the UBR5 promoter region based on JASPAR database analysis; (J) ChIP‐PCR analysis showing HIF1A enrichment at the UBR5 promoter region; (K) dual‐luciferase reporter assay evaluating the regulatory effect of HIF1A on UBR5 promoter activity; (L, M) RT‐qPCR analysis of HIF1A and UBR5 mRNA expression levels after shHIF1A knockdown in SKOV3 and OVCAR‐3 cells; (N, O) RT‐qPCR analysis of UBR5 mRNA expression levels in SKOV3 and OVCAR‐3 cells under normoxia, hypoxia, hypoxia + shNC, and hypoxia + shHIF1A conditions. Data are presented as mean ± SD; all cell experiments were performed in triplicate. *** p < 0.001, analyzed by ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Knockdown, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: GEM regulates LATS2 ubiquitination and YAP/TAZ phosphorylation via the hypoxia‐inducible factor 1‐alpha/UBR5 axis. (A) Western blot (WB) analysis of Hippo pathway‐related proteins in SKOV3 cells under hypoxic conditions; (B) WB analysis of Hippo pathway‐related proteins in OVCAR‐3 cells under hypoxic conditions; (C) co‐IP analysis showing the interaction between LATS2 and UBR5 in SKOV3 cells; (D) Co‐IP analysis showing the interaction between LATS2 and UBR5 in OVCAR‐3 cells; (E) ubiquitination assay measuring LATS2 ubiquitination levels in SKOV3 cells treated with 20 μM GEM for 48 h; (F) ubiquitination assay measuring LATS2 ubiquitination levels in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (G) WB analysis of in vitro ubiquitination to evaluate UBR5‐mediated LATS2 ubiquitination; (H) WB analysis of Hippo pathway‐related proteins in SKOV3 cells treated with 20 μM GEM for 48 h; (I) WB analysis of Hippo pathway‐related proteins in OVCAR‐3 cells treated with 20 μM GEM for 48 h. Results are presented as mean ± standard deviation. All experiments were repeated three times. *** p < 0.001, using ANOVA and Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Standard Deviation, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: Investigation of GEM's regulation of the Hippo pathway in ovarian cancer inhibition. (A) Western blot (WB) analysis of YAP1 and FGFR1 expression levels in SKOV3 cells treated with 20 μM GEM for 48 h; (B) WB analysis of YAP1 and FGFR1 expression levels in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (C) CCK8 assay measuring cell viability in SKOV3 cells treated with 20 μM GEM for 48 h; (D) CCK8 assay measuring cell viability in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (E) wound healing assay showing migration rates of SKOV3 cells treated with 20 μM GEM for 48 h (scale bar: 100 μm); (F) wound healing assay showing migration rates of OVCAR‐3 cells treated with 20 μM GEM for 48 h (scale bar: 100 μm); (G) transwell assay showing invasion ability of SKOV3 cells treated with 20 μM GEM for 48 h (scale bar: 50 μm); (H) transwell assay showing invasion ability of OVCAR‐3 cells treated with 20 μM GEM for 48 h (scale bar: 50 μm). Results are presented as mean ± standard deviation. Cell experiments were repeated 3 times; *** p < 0.001, using ANOVA and Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Inhibition, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Assay, Standard Deviation, Comparison