Journal: iScience

Article Title: ISG12a promotes immunotherapy of HBV-associated hepatocellular carcinoma through blocking TRIM21/AKT/β-Catenin/PD-L1 axis

doi: 10.1016/j.isci.2024.109533

Figure Lengend Snippet: Figure 1. Influence of HBV infection on malignant phenotypes of HLCZ01 cells (A) Immunoblots for p-STAT1, STAT1, p-STAT2, STAT2 levels in lysates of HLCZ01 cells with or without HBV infection. Cells were infected with NDV for 16 h, and the multiplicity of infection (MOI) of NDV was 0.2. ND, not detected. (B) Immunoblots for PD-L1, p-AKT, AKT, p-mTOR, and mTOR levels in lysates of HLCZ01 cells with or without HBV infection. (C) Flow cytometry analysis for surface PD-L1 level on HLCZ01 cells with or without HBV infection. (D) Proliferation of HLCZ01 cells with or without HBV infection. Cell proliferation was determined by counting using a blood counting chamber. (E) Immunofluorescence staining for F-actin level in HLCZ01 cells with or without HBV infection. Magnification: 6003, and scale bar: 20 mm. (F) Tumor formation of HLCZ01 cells with or without HBV infection in NCG mice (n = 5 or 6). Minimum scale of the ruler: 1 mm. Experiments were independently replicated at least two times with similar results. Data were analyzed by one-way ANOVA (A) or unpaired two-sided Student’s t tests (B–D and F), and are presented as mean G SEM with three replicate experiments (A and B) or at least three biological replicates (C, D, and F). **p < 0.01 and ***p < 0.001.

Article Snippet: Following antibodies were used for immunoblots and co-IP: ISG12a (1:1000, Cat# SAB1408588, Sigma-Aldrich), GAPDH (1:2000, clone 6C5, Cat#MAB374,MerckMillipore, Darmstadt, Germany), Flag (1:5000, clone M2, Cat# F3165, Sigma-Aldrich), V5 (1:2000, Cat# R960-25, Thermo Fisher Scientific), HA (1:1000, clone C29F4, Cat# 3724S, Cell Signaling Technology, Danvers, MA, USA), b-Catenin (1:1000, clone D10A8, Cat# 8480S, Cell Signaling Technology), p-b-Catenin (1:1000, clone D8E11, Cat# 5651S, Cell Signaling Technology), PD-L1 (1:250, Cat# 17952-1-AP, Proteintech, Rosemont, IL, USA), AKT (1:1000, clone C67E7, Cat# 4691S, Cell Signaling Technology), p-AKT (1:1000, clone D9E, Cat# 4060S, Cell Signaling Technology), mTOR (1:500, clone 30, Cat# sc-517464, Santa Cruz Technology, Dallas, TX, USA), p-mTOR (1:500, clone 59, Cat# sc-293133, Santa Cruz Technology), HBsAg (1:250, clone 1025, Cat# sc-53300, Santa Cruz Technology), HBcAg (1:250, clone C1-5, Cat# sc-23945, Santa Cruz Technology), b-actin (1:5000, cloneAC-74, Cat# A5316, Sigma-Aldrich), InVivoSIM anti-human PD-1 (NivolumabBiosimilar) (1:500, cloneNivolumab, Cat# SIM0003, Bio X Cell, NH, USA), goat anti-mouse IgG (HRP-linked) (1:5000, Cat# AP124P, Merck Millipore), goat anti-rabbit IgG (HRP-linked) (1:5000, 18 iScience 27, 109533, April 19, 2024 Cat# AP132P, Merck Millipore), and HRP-conjugated goat anti-human IgG (1:500, Cat# D110150, Sangon Biotech).

Techniques: Infection, Western Blot, Flow Cytometry, Staining

Journal: iScience

Article Title: ISG12a promotes immunotherapy of HBV-associated hepatocellular carcinoma through blocking TRIM21/AKT/β-Catenin/PD-L1 axis

doi: 10.1016/j.isci.2024.109533

Figure Lengend Snippet: Figure 2. Inhibitory effect of ISG12a on PD-L1 expression and cancer phenotypes of HBV-infected HLCZ01 cells (A) Immunoblots for ISG12a level in HLCZ01 cells with or without HBV infection. (B) Immunoblots for PD-L1, p-AKT, AKT, p-mTOR, mTOR, and ISG12a levels in lysates of HBV-infected HLCZ01 cells with ISG12a knockdown. (C) ELISA for HBsAg level in supernatants of HBV-infected HLCZ01 cells with ISG12a knockdown. (D) Flow cytometry analysis for surface PD-L1 level on HBV-infected HLCZ01 cells with ISG12a knockdown. (E) Proliferation of HBV-infected HLCZ01 cells with ISG12a knockdown. Cell proliferation was determined by counting using a blood counting chamber. (F) Immunofluorescence staining for F-actin level in HBV-infected HLCZ01 cells with ISG12a knockdown. Magnification: 6003, and scale bar: 20 mm. (G) Immunoblots for PD-L1, p-AKT, AKT, p-mTOR, mTOR, and Flag levels in HBV-infected HLCZ01 cells. The stably infected cells using lentivirus for silencing ISG12a expression were transfected with vector control or Flag-ISG12a plasmid for 72 h. (H) Immunofluorescence staining for F-actin level in HBV-infected HLCZ01 cells with the overexpression of ISG12a. Magnification: 6003, and scale bar: 20 mm.

Article Snippet: Following antibodies were used for immunoblots and co-IP: ISG12a (1:1000, Cat# SAB1408588, Sigma-Aldrich), GAPDH (1:2000, clone 6C5, Cat#MAB374,MerckMillipore, Darmstadt, Germany), Flag (1:5000, clone M2, Cat# F3165, Sigma-Aldrich), V5 (1:2000, Cat# R960-25, Thermo Fisher Scientific), HA (1:1000, clone C29F4, Cat# 3724S, Cell Signaling Technology, Danvers, MA, USA), b-Catenin (1:1000, clone D10A8, Cat# 8480S, Cell Signaling Technology), p-b-Catenin (1:1000, clone D8E11, Cat# 5651S, Cell Signaling Technology), PD-L1 (1:250, Cat# 17952-1-AP, Proteintech, Rosemont, IL, USA), AKT (1:1000, clone C67E7, Cat# 4691S, Cell Signaling Technology), p-AKT (1:1000, clone D9E, Cat# 4060S, Cell Signaling Technology), mTOR (1:500, clone 30, Cat# sc-517464, Santa Cruz Technology, Dallas, TX, USA), p-mTOR (1:500, clone 59, Cat# sc-293133, Santa Cruz Technology), HBsAg (1:250, clone 1025, Cat# sc-53300, Santa Cruz Technology), HBcAg (1:250, clone C1-5, Cat# sc-23945, Santa Cruz Technology), b-actin (1:5000, cloneAC-74, Cat# A5316, Sigma-Aldrich), InVivoSIM anti-human PD-1 (NivolumabBiosimilar) (1:500, cloneNivolumab, Cat# SIM0003, Bio X Cell, NH, USA), goat anti-mouse IgG (HRP-linked) (1:5000, Cat# AP124P, Merck Millipore), goat anti-rabbit IgG (HRP-linked) (1:5000, 18 iScience 27, 109533, April 19, 2024 Cat# AP132P, Merck Millipore), and HRP-conjugated goat anti-human IgG (1:500, Cat# D110150, Sangon Biotech).

Techniques: Expressing, Infection, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Stable Transfection, Transfection, Plasmid Preparation, Control, Over Expression

Journal: Cancer cell international

Article Title: Proapoptotic role of CDK1 in overcoming paclitaxel resistance in ovarian cancer cells in response to combined treatment with paclitaxel and duloxetine.

doi: 10.1186/s12935-024-03607-8

Figure Lengend Snippet: Fig. 6 Treatment with duloxetine overcomes paclitaxel resistance in SKOV3-TR cells via phosphorylation of the antiapoptotic Bcl-2 family in a CDK1- dependent manner. (A) SKOV3-TR cells were treated with 100 nM paclitaxel and 15 µM duloxetine for 48 h. Cell viability was measured by MTT assay. (B) The cells were treated with 100 nM paclitaxel and 15 µM duloxetine for 12 h. The expression levels of mTOR, S6K and S6 were detected by immunoblot analysis. (C) The cells were treated with 100 nM paclitaxel and 15 µM duloxetine in combination with 10 µM Ro-3306 for 48 h. Apoptotic cell death was determined by Annexin V-FITC/PI staining using flow cytometry. A statistical graph of apoptotic cells was shown. (D, E) The cells were treated with 100 nM paclitaxel and 15 µM duloxetine in combination with 10 µM Ro-3306 for 12 h. (D) The expression levels of CDK1 and cyclin B1 were detected by im munoblot analysis. (E) The expression levels of apoptotic cleaved PARP and the antiapoptotic Bcl-2 family were detected by immunoblot analysis. The relative optical densities were quantified using ImageJ software. The data are presented as the mean ± SD of the percentage or the fold change relative to the control (n = 3, * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significantly different). PTX, paclitaxel; DLX, duloxetine; RO, Ro-3306

Article Snippet: The following antibodies were used: Cleaved PARP (#9541, Cell Signaling Technology), p-Bcl-2 (S70) (#2827, Cell Signaling Technology), Bcl-2 (#A19693, ABclonal, Wuhan, Hubei, China), p-BclxL (S62) (#AP0314, ABclonal), Bcl-xL (#2764, Cell Signaling Technology), p-CDK1 (Y15) (#9111, Cell Signaling Technology), p-CDK1 (T161) (#9114, Cell Signaling Technology), CDK1 (#9116, Cell Signaling Technology), Cyclin B1 (#sc-245, Santa Cruz Biotechnology), COX IV (#4850, Cell Signaling Technology), p-mTOR (S2448) (#2971, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), p-S6K (T389) (#9205, Cell Signaling Technology), S6K (#9202, Cell Signaling Technology), p-S6 (S240/244) (#5364, Cell Signaling Technology), S6 (#2217, Cell Signaling Technology), β-Actin (#A5316, Sigma‒Aldrich), Goat anti-Rabbit IgG-heavy and light chain Antibody HRP Conjugated (#A120-101P, Bethyl Laboratories Inc., Montgomery, TX, USA), and Goat Anti-Mouse IgG (H + L) HRP (#A1012S, ACE Biolabs, Foshan, Guangdong, China).

Techniques: Phospho-proteomics, MTT Assay, Expressing, Western Blot, Staining, Flow Cytometry, Software, Control

Journal: Cancer cell international

Article Title: Proapoptotic role of CDK1 in overcoming paclitaxel resistance in ovarian cancer cells in response to combined treatment with paclitaxel and duloxetine.

doi: 10.1186/s12935-024-03607-8

Figure Lengend Snippet: Fig. 7 Treatment with duloxetine enhances sensitivity of paclitaxel in parental ovarian cancer cells. (A) HEYA8 cells were treated with 5 nM paclitaxel and 15 µM duloxetine for 48 h. Cell viability was measured by MTT assay. (B) HEYA8 were treated with 5 nM paclitaxel and 15 µM duloxetine for 12 h. The expression levels of mTOR, S6K and S6 were detected by immunoblot analysis. (C) HEYA8 were treated with 5 nM paclitaxel and 15 µM duloxetine in combination with 10 µM Ro-3306 for 48 h. Apoptotic cell death was determined by Annexin V-FITC/PI staining using flow cytometry. A statistical graph of apoptotic cells was shown. (D) HEYA8 were treated with 5 nM paclitaxel and 15 µM duloxetine in combination with 10 µM Ro-3306 for 12 h. The expres sion levels of CDK1 and cyclin B1 were detected by immunoblot analysis. The relative optical densites was quantified using ImageJ software. The data are presented as the mean ± SD of the percentage or the fold change relative to the control (n = 3,; ** p < 0.01; *** p < 0.001; ns, not significantly different). (E) Proposed mechanism of duloxetine-mediated sensitization of ovarian cancer cells to paclitaxel. PTX, paclitaxel; DLX, duloxetine; RO, Ro-3306

Article Snippet: The following antibodies were used: Cleaved PARP (#9541, Cell Signaling Technology), p-Bcl-2 (S70) (#2827, Cell Signaling Technology), Bcl-2 (#A19693, ABclonal, Wuhan, Hubei, China), p-BclxL (S62) (#AP0314, ABclonal), Bcl-xL (#2764, Cell Signaling Technology), p-CDK1 (Y15) (#9111, Cell Signaling Technology), p-CDK1 (T161) (#9114, Cell Signaling Technology), CDK1 (#9116, Cell Signaling Technology), Cyclin B1 (#sc-245, Santa Cruz Biotechnology), COX IV (#4850, Cell Signaling Technology), p-mTOR (S2448) (#2971, Cell Signaling Technology), mTOR (#2983, Cell Signaling Technology), p-S6K (T389) (#9205, Cell Signaling Technology), S6K (#9202, Cell Signaling Technology), p-S6 (S240/244) (#5364, Cell Signaling Technology), S6 (#2217, Cell Signaling Technology), β-Actin (#A5316, Sigma‒Aldrich), Goat anti-Rabbit IgG-heavy and light chain Antibody HRP Conjugated (#A120-101P, Bethyl Laboratories Inc., Montgomery, TX, USA), and Goat Anti-Mouse IgG (H + L) HRP (#A1012S, ACE Biolabs, Foshan, Guangdong, China).

Techniques: MTT Assay, Expressing, Western Blot, Staining, Flow Cytometry, Software, Control

Journal: NPJ precision oncology

Article Title: Taurine and proline promote lung tumour growth by co-regulating Azgp1/mTOR signalling pathway.

doi: 10.1038/s41698-025-00872-2

Figure Lengend Snippet: Fig. 7 | Azgp1/mTOR axis is identified as an under-reported pathway for taurine and proline in promoting lung cancer progression. Taurine and proline may inhibit AZGP1 function, triggering mTOR pathway activation and lipid metabolism disorder that ultimately promote lung cancer progression.

Article Snippet: The following antibodies were used in this experiment: β-Actin (Cell Signalling Technology (CST), #8457), p-mTOR (Santa Cruz, sc-293133), mTOR (CST, #2983), Azgp1 (ZAP) (Santa Cruz, sc-21720).

Techniques: Activation Assay