Journal: EMBO Molecular Medicine

Article Title: AI-identified CD133-targeting natural compounds demonstrate differential anti-tumor effects and mechanisms in pan-cancer models

doi: 10.1038/s44321-025-00308-1

Figure Lengend Snippet: ( A ) Expression level analysis of CD133 in pan-cancer from the TCGA database. Expression-level analysis of CD133 in CRC from the TCGA database ( B ) and GSE21815 dataset from the GEO database ( C ). Kaplan–Meier estimator of overall survival rate based on CD133 expression from the TCGA database ( D ) and GSE30378 dataset from the GEO database ( E ). ( F ) Quantitative IHC results for CD133 expressed in pan-cancer patients as average optical density (AOD) values. ( G ) IHC staining of CRC patient’s tumor tissue section and normal tissue section on CD133. ( H ) Quantitative IHC results for CD133 expressed in CRC patients as AOD values. Scale bar = 50 μm. Student’s t test. Exact P values are presented in Appendix Table . Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. .

Article Snippet: The following primary antibodies (1:1000) were used: Caspase-1 (A0964), GSDMD (A20197), IL-1β (A16288), Caspase-3 (A19654), Bax (A12009), Bcl-2 (A19693), β-actin (AC038), p-mTOR (AP0978), mTOR (A11355), p-PI3K (AP0427), PI3K (A4992), p-Akt (AP1332), Akt (A22770), P62 (A11483), LC3B (A11282), p-P65 (AP1294), P65 (A22331), β-catenin (A11512), Dvl2 (A23686), Naked-1 (A17799), p-GSK3β (AP1341), GSK3β (A2081), LEF1 (A23458), TCF1/7 (A20835), CD133 (12711), N-cadherin (A0433), E-cadherin (A24874), Vimentin (A19607), and Snail (A11794), which were bought from ABclonal Technology Co. Ltd.,

Techniques: Expressing, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: AI-identified CD133-targeting natural compounds demonstrate differential anti-tumor effects and mechanisms in pan-cancer models

doi: 10.1038/s44321-025-00308-1

Figure Lengend Snippet: ( A ) The IHC staining of CD133 in various cancer, including liver cancer, lung cancer, colorectal cancer, and gastric cancer. Scale bar, 50 μm. ( B , C ) Expression level of signaling pathway in WT and shCD133 HCT116 cell lines. ( B ) PI3K–AKT pathway, ( C ) Wnt-β-catenin pathway ( D , E ) Cell viability assay results showing the cytotoxicity of PP10 and PP24 in FHC cells. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 when compared with control group.

Article Snippet: The following primary antibodies (1:1000) were used: Caspase-1 (A0964), GSDMD (A20197), IL-1β (A16288), Caspase-3 (A19654), Bax (A12009), Bcl-2 (A19693), β-actin (AC038), p-mTOR (AP0978), mTOR (A11355), p-PI3K (AP0427), PI3K (A4992), p-Akt (AP1332), Akt (A22770), P62 (A11483), LC3B (A11282), p-P65 (AP1294), P65 (A22331), β-catenin (A11512), Dvl2 (A23686), Naked-1 (A17799), p-GSK3β (AP1341), GSK3β (A2081), LEF1 (A23458), TCF1/7 (A20835), CD133 (12711), N-cadherin (A0433), E-cadherin (A24874), Vimentin (A19607), and Snail (A11794), which were bought from ABclonal Technology Co. Ltd.,

Techniques: Immunohistochemistry, Expressing, Viability Assay, Control

Journal: EMBO Molecular Medicine

Article Title: AI-identified CD133-targeting natural compounds demonstrate differential anti-tumor effects and mechanisms in pan-cancer models

doi: 10.1038/s44321-025-00308-1

Figure Lengend Snippet: ( A ) Workflow detailing the steps involved in data preparation, featurization, and the Transformer module application. ( B ) Detailed process of the Transformer-based algorithm for compound-protein interaction prediction. ( C ) Brief workflow of the compound screening process. ( D ) MTT assay results showing the cytotoxicity of CL6, PP9, PP10, PP18, PP22, and PP24 on HCT116 cells. ( E , F ) Molecular docking analysis of PP10, PP24 with CD133. Hydrophobic interactions are depicted in dotted lines, and hydrogen bonds are depicted in blue straight lines. ( G , H ) SPR results of the binding interactions between PP10, PP24, and CD133. ( I , J ) CETSA results showing the thermal stability of CD133 in the presence of PP10 and PP24. ( K ) Expression level of CD133 in wild-type and shCD133 HCT116 cell lines. ( L ) Cell viability assay results showing the cytotoxicity of PP10 and PP24 in shCD133-HCT116 cells. Student’s t test. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 when compared with control group. .

Article Snippet: The following primary antibodies (1:1000) were used: Caspase-1 (A0964), GSDMD (A20197), IL-1β (A16288), Caspase-3 (A19654), Bax (A12009), Bcl-2 (A19693), β-actin (AC038), p-mTOR (AP0978), mTOR (A11355), p-PI3K (AP0427), PI3K (A4992), p-Akt (AP1332), Akt (A22770), P62 (A11483), LC3B (A11282), p-P65 (AP1294), P65 (A22331), β-catenin (A11512), Dvl2 (A23686), Naked-1 (A17799), p-GSK3β (AP1341), GSK3β (A2081), LEF1 (A23458), TCF1/7 (A20835), CD133 (12711), N-cadherin (A0433), E-cadherin (A24874), Vimentin (A19607), and Snail (A11794), which were bought from ABclonal Technology Co. Ltd.,

Techniques: MTT Assay, Binding Assay, Expressing, Viability Assay, Control

Journal: EMBO Molecular Medicine

Article Title: AI-identified CD133-targeting natural compounds demonstrate differential anti-tumor effects and mechanisms in pan-cancer models

doi: 10.1038/s44321-025-00308-1

Figure Lengend Snippet: PP10 and PP24 inhibited the cell viability of colorectal cancer organoids ( A ), lung cancer organoids ( B ) and liver cancer organoids ( C ) ( N = 3). ( D ) Expression level of CD133 in pan-cancer patient-derived organoids, namely, Colorectal cancer, Lung cancer, Liver cancer, Thyroid cancer, and Breast cancer. Scale bar = 50 μm. one-way ANOVA test. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, when compared with control group.

Article Snippet: The following primary antibodies (1:1000) were used: Caspase-1 (A0964), GSDMD (A20197), IL-1β (A16288), Caspase-3 (A19654), Bax (A12009), Bcl-2 (A19693), β-actin (AC038), p-mTOR (AP0978), mTOR (A11355), p-PI3K (AP0427), PI3K (A4992), p-Akt (AP1332), Akt (A22770), P62 (A11483), LC3B (A11282), p-P65 (AP1294), P65 (A22331), β-catenin (A11512), Dvl2 (A23686), Naked-1 (A17799), p-GSK3β (AP1341), GSK3β (A2081), LEF1 (A23458), TCF1/7 (A20835), CD133 (12711), N-cadherin (A0433), E-cadherin (A24874), Vimentin (A19607), and Snail (A11794), which were bought from ABclonal Technology Co. Ltd.,

Techniques: Expressing, Derivative Assay, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 6. Artesunate inhibited cancer stem cell–like properties of NSCLC (A-C) Artesunate decreased mammosphere formation in the three NSCLC cells. H1975, LLC, and H460 cells were plated into ultra-low attachment 6-well plates and treated with ART at the indicated concentrations for 72 h. Representative images were taken with microscope on days 7. (D-F) Quantification of mammosphere formation with the three NSCLC cells treated with DMSO or different concentrations of ART. (G-I) H1975, LLC, and H460 cells were treated with the indicated concentrations of ART for 48 h, and the mRNA levels of OCT4, Nestin, and CD133 were detected by quantitative RT-PCR analysis. Each experiment was performed in triplicate. Data are shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001 vs. the control group). (J-L) The protein levels of OCT4, Nestin, and CD133 in ART-treated and DMSO-treated NSCLC cells. Gapdh was used as a loading control. The results shown are representative of at least three independent experiments.

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

Techniques: Microscopy, Quantitative RT-PCR, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 2. Artesunate inhibited TAZ/PD-L1 signaling in NSCLC cells. (A, B) H1975 and H460 cells were treated with the indicated concentrations of ART for 48 h, and the mRNA levels of TAZ, ANKRD1, and CD274 were detected by quantitative RT-PCR anal ysis. Each experiment was performed in triplicate. Data are shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001 vs. control). (C, D) ART reduced the protein expression of TAZ, ANKRD1, and PD-L1 in the human NSCLC cells. H1975 and H460 cells were treated with the indicated con centrations of ART for 48 h, and the protein levels of TAZ, ANKRD1, and PD-L1 were detected by western blot analysis. Gapdh was used as a loading control. The results shown are representative of at least three indepen dent experiments. (E-H) Immunostaining of TAZ and PD-L1 (green) on human NSCLC cells treated with or without the indicated concentrations of ART for 48 h. The nuclei were stained with DAPI (blue). Scale bar, 10 µm.

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

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

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 7. Artesunate and gefitinib in combination exerted enhanced inhibitory activity against gefitinib-resistant NSCLC cells in vivo. H1975 cells grown in BALB/c nude mice as xenografted tumors were treated with vehicle, gefitinib (GEF) alone (50 mg/kg/day, og), artesunate (ART) alone (40 mg/kg/day, ip), or the combi nation of gefitinib with artesunate. (A, B) Tumor sizes and mouse body weights were measured every 3 days. (C, D) At the end of treatments, tumors in each group were also collected, weighed, and photographed. The data in each group are the mean ± SD of 5 tumors from 5 mice. (E) Representative IHC staining of TAZ, Ki67, and ANKRD1 in sections of the indicated tumor xenografts (Scale bars: 50 µm).

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

Techniques: Activity Assay, In Vivo, Immunohistochemistry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 2. Artesunate inhibited TAZ/PD-L1 signaling in NSCLC cells. (A, B) H1975 and H460 cells were treated with the indicated concentrations of ART for 48 h, and the mRNA levels of TAZ, ANKRD1, and CD274 were detected by quantitative RT-PCR anal ysis. Each experiment was performed in triplicate. Data are shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001 vs. control). (C, D) ART reduced the protein expression of TAZ, ANKRD1, and PD-L1 in the human NSCLC cells. H1975 and H460 cells were treated with the indicated con centrations of ART for 48 h, and the protein levels of TAZ, ANKRD1, and PD-L1 were detected by western blot analysis. Gapdh was used as a loading control. The results shown are representative of at least three indepen dent experiments. (E-H) Immunostaining of TAZ and PD-L1 (green) on human NSCLC cells treated with or without the indicated concentrations of ART for 48 h. The nuclei were stained with DAPI (blue). Scale bar, 10 µm.

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

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

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 3. Artesunate suppressed TAZ/PD-L1–induced T-cell growth inhibition and enhanced the killing activity of Jurkat T-cells on NSCLC cells. (A) PD-1 expression in Jurkat T-cells was stimulated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL phytohemagglutinin (PHA) for 48 h. (B) Artesunate increased T-cell growth in co-culture experiments. T-cell proliferation in co-culture system was determined by CCK-8 assay. (C) Artesunate suppressed the caspase-3/- 7 activity of Jurkat T cells in co-culture experiments. Caspase-3/-7 activity is expressed as the percentage of the DMSO-treated cells. (D) Representative images of annexin-V-FITC/PI assay. Artesunate suppressed T-cell apoptosis in co-culture experiments. (E) Statistical analysis of T-cell apoptosis. (F) Representative histograms of relative surviving cell intensity. Activated Jurkat T cell and ART-pretreated H1975 cells were co-cultured in 6-well plates for 2 days. SRB staining was used to monitor the surviving cancer cells. Data were presented as mean ± SD, n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

Techniques: Inhibition, Activity Assay, Expressing, Co-Culture Assay, CCK-8 Assay, Cell Culture, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 9. Artesunate targeted the TAZ–TEAD complex and promoted TAZ degradation in a proteasome-dependent manner (A) MCF10A-TAZ-S89A cells were treated with the indicated concentrations of ART for 48 h, and the protein levels of TAZ and PD-L1 were detected by western blot analysis. Gapdh was used as a loading control. The results shown are representative of at least three independent experiments. (B) H1975 cells were treated with ART or a combination of ART and CHX for the indicated time. TAZ protein expression levels were detected by western blot analysis. The results shown are representative of three independent experiments. (C) H1975 cells were treated with ART or a combination of ART and MG132 for the indicated time and then analyzed with Western blotting to detect TAZ abundance. (D) Representative images of temperature-dependent cellular thermal shift assay. H1975 cell lysates were heated at different temperatures (45–60 ◦C) for 4 min. The supernatants of the heated samples were analyzed by western blotting. (E) Molecular docking predicted the potential binding of artesunate to TAZ-TEAD4 complex, and artesunate (Gray); the active sites of TAZ are LYS-46 (Blue), SER-41 (Blue); and the hydrogen bonds are yellow in 3D model, and the hydrogen bonds are green in 2D model.

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

Techniques: Western Blot, Control, Expressing, Thermal Shift Assay, Binding Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Artesunate promoted anti-tumor immunity and overcame EGFR-TKI resistance in non-small-cell lung cancer by enhancing oncogenic TAZ degradation.

doi: 10.1016/j.biopha.2022.113705

Figure Lengend Snippet: Fig. 6. Artesunate inhibited cancer stem cell–like properties of NSCLC (A-C) Artesunate decreased mammosphere formation in the three NSCLC cells. H1975, LLC, and H460 cells were plated into ultra-low attachment 6-well plates and treated with ART at the indicated concentrations for 72 h. Representative images were taken with microscope on days 7. (D-F) Quantification of mammosphere formation with the three NSCLC cells treated with DMSO or different concentrations of ART. (G-I) H1975, LLC, and H460 cells were treated with the indicated concentrations of ART for 48 h, and the mRNA levels of OCT4, Nestin, and CD133 were detected by quantitative RT-PCR analysis. Each experiment was performed in triplicate. Data are shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001 vs. the control group). (J-L) The protein levels of OCT4, Nestin, and CD133 in ART-treated and DMSO-treated NSCLC cells. Gapdh was used as a loading control. The results shown are representative of at least three independent experiments.

Article Snippet: The following antibodies were used in this study: TAZ (#A8202; ABclonal, Wuhan, China), GAPDH (#2118; Cell Signaling Technology, Danvers, MA, USA), ANKRD1 (#A6192; ABclonal), PD-L1 (#A1645; ABclonal), Nestin (#A11861; ABclonal), CD133 (#A0219; ABclonal), OCT4 (#A7920; ABclonal), horseradish peroxidase (HRP)-conjugated anti-mouse (#1706516; BioRad Laboratories), and HRP-conjugated anti-rabbit (#1706515; BioRad Laboratories).

Techniques: Microscopy, Quantitative RT-PCR, Control

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) interacts with protein arginine methyltransferase 2 (PRMT2) and maintains stability of PRMT2. (A) Mass spectrometry analysis identified PRMT2 bound with PADI4. (B) Immunostaining of ECA109 cells transfected with PADI4‐HA and PRMT2‐Flag was performed using anti‐HA and anti‐Flag antibodies, with confocal microscopy revealing the subcellular localization of PADI4‐Flag (green), PRMT2 (red) and DAPI (blue, nucleus marker). (C, D) Endogenous cimmunoprecipitation was used to study the interaction between PADI4 and PRMT2 in ECA109 and KYSE‐150 cells after transformed into plasmid of PADI4‐HA. (E, F) PRMT2 protein level in PADI4‐knockdown cell lines. (G, H) PRMT2 protein level in PADI4‐upregulated cell lines. (I) PRMT2 protein degradation levels were measured in treat with cycloheximide (CHX) (10 µg/mL) over specified time intervals in PADI4‐KO ECA109 cells. (J) Lysates from cells co‐transfected with His‐Ub, Flag‐PRMT2 and PADI4‐HA underwent immunoprecipitation with a Flag antibody, followed by immunoblotting with an anti‐His antibody in HEK 293T cells. DAPI, 4′,6‐Diamidino‐2‐phenylindole.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Mass Spectrometry, Immunostaining, Transfection, Confocal Microscopy, Marker, Transformation Assay, Plasmid Preparation, Knockdown, Immunoprecipitation, Western Blot

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) citrullinates protein arginine methyltransferase 2 (PRMT2) to maintain the stability of PRMT2. (A) PADI4‐HA overexpression was immunoprecipitated using Flag‐PRMT2 antibodies, and immunoblotted with modified‐citrulline antibodies. (B) Recombinant PADI4 was immunoprecipitated with a Flag‐PRMT2 antibody in calcium presence, and reaction products were analysed by western blot with modified‐citrulline antibody. (C) The amino acid sequence of PRMT2 in different species is highly conserved at R312 and R397. (D) Two PRMT2 mutant plasmids and transfected into HEK 293T cells to perform immunoprecipitation experiments to investigate citrullination. (E) ECA109 cells were analysed by western blot after 8‐h treatment with varying doses of BB‐Cl‐Amidine. (F) In ECA109 cells treated with GSK484, PRMT2's half‐life was shown by using cycloheximide (CHX) to block protein synthesis. (G) Immunoprecipitating PRMT2 with an anti‐PRMT2‐Flag antibody and probing for polyubiquitination with an anti‐ubiquitin antibody revealed a significant increase in polyubiquitinated PRMT2 protein after GSK484 treatment. (H, I) KYSE150 (H) and ECA109 (I) cells were analysed of cancer stem cell (CSC) markers by western blot after 24‐h treatment with varying doses of GSK484.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Over Expression, Immunoprecipitation, Modification, Recombinant, Western Blot, Sequencing, Mutagenesis, Transfection, Blocking Assay, Ubiquitin Proteomics

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) acts as a positive regulator of inhibitors of DNA binding (IDs). (A, B) The mRNA and protein levels of cancer stem cell (CSC) markers in PRMT2 knockdown ECA109 cells were also assessed by qPCR and western blot. (C) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of CSC markers were measured using western blot. (D, E) The mRNA and protein levels of ID1, ID2 and ID3 in PRMT2 knockdown ECA109 cells were assessed using qPCR and western blot. (F) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of ID1, ID2 and ID3 were measured using western blot. (G, H) The mRNA and protein levels of ID1, ID2 and ID3 in peptidylarginine deiminase 4 (PADI4) knockdown and ECA109 cells were assessed using real‐time PCR and western blot. (I) The protein levels of ID1, ID2 and ID3 in ECA109 cells transfected with PADI4 plasmids were measured. (J) H3R8me2a expression was analysed via western blot in PADI4‐transfected ECA109 cells. (K, L) ChIP assay revealed H3R8me2a enrichment at the human ID1 and ID2 promoter in ECA109 cells. (M, N) Cell viability was assessed using a CCK8 assay after 48‐h treatment with cisplatin in ECA109 (M) and KYSE150 cells (N). (O) PRMT2‐KO cells showed reduced sphere formation in ECA109 and KYSE150 cells. (P, Q) GSK484 reduction in the expression levels of ID1, ID2 and ID3 with different concentration in ECA109 and KYSE150 cells. (R, S) Limiting dilution assays showing the self‐renewing capacity of PRMT2 downregulated in ECA109 (R) and KYSE150 (S) CD133‐positive cells. (T) PRMT2 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (U) Immunihistochemical (IHC) analysis comparing PRMT2 expression in oesophageal squamous cell carcinoma (OSCC) patients tumour tissues to adjacent normal oesophageal tissues. (V) Correlation between PADI4 and PRMT2 in OSCC patients tumour tissues.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Binding Assay, Knockdown, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, CCK-8 Assay, Concentration Assay, Colony Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: USP7 induces stability of protein arginine methyltransferase 2 (PRMT2) expression and spheroid formation of cancer stem cell (CSC). (A) Mass spectrometry analysis identified PRMT2 as a prospective target of USP7. (B) Conducted endogenous immunoprecipitation to study USP7 and PRMT2 interaction in ECA109 cells after transformed into plasmid of PRMT2‐Flag. (C) USP7‐KO decreased the expression of PRMT2 in ECA109 cell line via western blot. (D) PRMT2 expression was analysed via western blot in ECA109 cells transfected with USP7‐HA. (E) qPCR showed USP7 knockdown significantly reduced Nanog and CD133 and mRNA levels in ECA109 cells. (F) Western blot analysis showed CSC markers decreased in USP7‐KO ECA109 cell. (G) FACS analysis revealed that USP7 knockdown significantly reduced CD44+ cell percentages in KYSE150 cells. (H) USP7 knockdown significantly impaired sphere formation in KYSE150 cells. (I) Western blots of USP7 knockout ECA109 cell treated with DMSO or MG132 (20 µM) for 5 h. (J) ECA109 cells with USP7 knockout were treated with cycloheximide (CHX) (10 µg/mL) for varying durations, and PRMT2 protein levels were assessed by western blotting. (K) HEK 293T cells co‐transfected with HA‐USP7, His‐Ub and PRMT2‐Flag were treated with MG132 (20 µM) for 5 h before harvesting to evaluate PRMT2 ubiquitination. (L, M) qPCR and western blot analysis of IDs from USP7 knockout ECA109 cells. (N) Analyse USP7 knockout cells expressing H3R8me2a. (O) Conduct western blot analysis on lysates with HA‐USP7 to assess H3R8me2a.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Expressing, Mass Spectrometry, Immunoprecipitation, Transformation Assay, Plasmid Preparation, Western Blot, Transfection, Knockdown, Knock-Out, Ubiquitin Proteomics

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) citrullinate disrupts the interaction between USP7 and PRMT2. (A) HEK 293T cells were co‐transfected with PRMT2‐Flag and PADI4‐HA plasmids, and USP7 levels were assessed via western blot. (B) Co‐transfect HEK 293T cells with PADI4‐Flag and USP7‐HA, treat with GSK484 (20 µM) for 24 h, and use western blot to evaluate PRMT2 levels. (C) HEK 293T cells were co‐transfected with PRMT2‐WT‐Flag, PRMT2‐R312E‐Flag and PRMT2‐R397E‐Flag plasmids, and USP7 levels were assessed via western blot. (D) HEK 293T cells were co‐transfected with HA‐USP7, His‐Ub, PRMT2‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, then treated with MG132 (20 µM) for 5 h before harvesting to assess PRMT2‐Flag ubiquitination levels. (E) HEK 293T cells were co‐transfected with HA‐USP7, PRMT2‐WT‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, and then use western blot to evaluate Flag levels. (F) Western blot was used to analyse ID1, ID2 and ID3 expression in HEK 293T cells transfected with PRMT2‐WT, PRMT2‐R397E and PRMT2‐R312E. (G) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate resistance of ECA109 cells to cisplatin. (H) Peptidylarginine deiminase 4 (PADI4) knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (I) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate the sphere formation. (J) PADI4 knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (K) Downregulation of PADI4 enhanced the resistance of ECA109 cells to cisplatin through CCK8 assay and PRMT2 restores this effect.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Knockdown, CCK-8 Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: USP7 induces stability of protein arginine methyltransferase 2 (PRMT2) expression and spheroid formation of cancer stem cell (CSC). (A) Mass spectrometry analysis identified PRMT2 as a prospective target of USP7. (B) Conducted endogenous immunoprecipitation to study USP7 and PRMT2 interaction in ECA109 cells after transformed into plasmid of PRMT2‐Flag. (C) USP7‐KO decreased the expression of PRMT2 in ECA109 cell line via western blot. (D) PRMT2 expression was analysed via western blot in ECA109 cells transfected with USP7‐HA. (E) qPCR showed USP7 knockdown significantly reduced Nanog and CD133 and mRNA levels in ECA109 cells. (F) Western blot analysis showed CSC markers decreased in USP7‐KO ECA109 cell. (G) FACS analysis revealed that USP7 knockdown significantly reduced CD44+ cell percentages in KYSE150 cells. (H) USP7 knockdown significantly impaired sphere formation in KYSE150 cells. (I) Western blots of USP7 knockout ECA109 cell treated with DMSO or MG132 (20 µM) for 5 h. (J) ECA109 cells with USP7 knockout were treated with cycloheximide (CHX) (10 µg/mL) for varying durations, and PRMT2 protein levels were assessed by western blotting. (K) HEK 293T cells co‐transfected with HA‐USP7, His‐Ub and PRMT2‐Flag were treated with MG132 (20 µM) for 5 h before harvesting to evaluate PRMT2 ubiquitination. (L, M) qPCR and western blot analysis of IDs from USP7 knockout ECA109 cells. (N) Analyse USP7 knockout cells expressing H3R8me2a. (O) Conduct western blot analysis on lysates with HA‐USP7 to assess H3R8me2a.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Expressing, Mass Spectrometry, Immunoprecipitation, Transformation Assay, Plasmid Preparation, Western Blot, Transfection, Knockdown, Knock-Out, Ubiquitin Proteomics

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) citrullinate disrupts the interaction between USP7 and PRMT2. (A) HEK 293T cells were co‐transfected with PRMT2‐Flag and PADI4‐HA plasmids, and USP7 levels were assessed via western blot. (B) Co‐transfect HEK 293T cells with PADI4‐Flag and USP7‐HA, treat with GSK484 (20 µM) for 24 h, and use western blot to evaluate PRMT2 levels. (C) HEK 293T cells were co‐transfected with PRMT2‐WT‐Flag, PRMT2‐R312E‐Flag and PRMT2‐R397E‐Flag plasmids, and USP7 levels were assessed via western blot. (D) HEK 293T cells were co‐transfected with HA‐USP7, His‐Ub, PRMT2‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, then treated with MG132 (20 µM) for 5 h before harvesting to assess PRMT2‐Flag ubiquitination levels. (E) HEK 293T cells were co‐transfected with HA‐USP7, PRMT2‐WT‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, and then use western blot to evaluate Flag levels. (F) Western blot was used to analyse ID1, ID2 and ID3 expression in HEK 293T cells transfected with PRMT2‐WT, PRMT2‐R397E and PRMT2‐R312E. (G) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate resistance of ECA109 cells to cisplatin. (H) Peptidylarginine deiminase 4 (PADI4) knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (I) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate the sphere formation. (J) PADI4 knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (K) Downregulation of PADI4 enhanced the resistance of ECA109 cells to cisplatin through CCK8 assay and PRMT2 restores this effect.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Knockdown, CCK-8 Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) acts as a positive regulator of inhibitors of DNA binding (IDs). (A, B) The mRNA and protein levels of cancer stem cell (CSC) markers in PRMT2 knockdown ECA109 cells were also assessed by qPCR and western blot. (C) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of CSC markers were measured using western blot. (D, E) The mRNA and protein levels of ID1, ID2 and ID3 in PRMT2 knockdown ECA109 cells were assessed using qPCR and western blot. (F) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of ID1, ID2 and ID3 were measured using western blot. (G, H) The mRNA and protein levels of ID1, ID2 and ID3 in peptidylarginine deiminase 4 (PADI4) knockdown and ECA109 cells were assessed using real‐time PCR and western blot. (I) The protein levels of ID1, ID2 and ID3 in ECA109 cells transfected with PADI4 plasmids were measured. (J) H3R8me2a expression was analysed via western blot in PADI4‐transfected ECA109 cells. (K, L) ChIP assay revealed H3R8me2a enrichment at the human ID1 and ID2 promoter in ECA109 cells. (M, N) Cell viability was assessed using a CCK8 assay after 48‐h treatment with cisplatin in ECA109 (M) and KYSE150 cells (N). (O) PRMT2‐KO cells showed reduced sphere formation in ECA109 and KYSE150 cells. (P, Q) GSK484 reduction in the expression levels of ID1, ID2 and ID3 with different concentration in ECA109 and KYSE150 cells. (R, S) Limiting dilution assays showing the self‐renewing capacity of PRMT2 downregulated in ECA109 (R) and KYSE150 (S) CD133‐positive cells. (T) PRMT2 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (U) Immunihistochemical (IHC) analysis comparing PRMT2 expression in oesophageal squamous cell carcinoma (OSCC) patients tumour tissues to adjacent normal oesophageal tissues. (V) Correlation between PADI4 and PRMT2 in OSCC patients tumour tissues.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Binding Assay, Knockdown, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, CCK-8 Assay, Concentration Assay, Colony Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) citrullinate disrupts the interaction between USP7 and PRMT2. (A) HEK 293T cells were co‐transfected with PRMT2‐Flag and PADI4‐HA plasmids, and USP7 levels were assessed via western blot. (B) Co‐transfect HEK 293T cells with PADI4‐Flag and USP7‐HA, treat with GSK484 (20 µM) for 24 h, and use western blot to evaluate PRMT2 levels. (C) HEK 293T cells were co‐transfected with PRMT2‐WT‐Flag, PRMT2‐R312E‐Flag and PRMT2‐R397E‐Flag plasmids, and USP7 levels were assessed via western blot. (D) HEK 293T cells were co‐transfected with HA‐USP7, His‐Ub, PRMT2‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, then treated with MG132 (20 µM) for 5 h before harvesting to assess PRMT2‐Flag ubiquitination levels. (E) HEK 293T cells were co‐transfected with HA‐USP7, PRMT2‐WT‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, and then use western blot to evaluate Flag levels. (F) Western blot was used to analyse ID1, ID2 and ID3 expression in HEK 293T cells transfected with PRMT2‐WT, PRMT2‐R397E and PRMT2‐R312E. (G) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate resistance of ECA109 cells to cisplatin. (H) Peptidylarginine deiminase 4 (PADI4) knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (I) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate the sphere formation. (J) PADI4 knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (K) Downregulation of PADI4 enhanced the resistance of ECA109 cells to cisplatin through CCK8 assay and PRMT2 restores this effect.

Article Snippet: Primary antibodies: PADI4 (17373‐1‐AP, Proteintech), PRMT2 (A5835, Abclonal), ID1 (YP‐Ab‐04970, UpingBio), ID2 (YP‐Ab‐07311, UpingBio), ID3 (YP‐Ab‐07306, UpingBio), H3R8me2a (A3157, Abclonal), GAPDH (A19056, Abclonal), β‐actin (AC026, Abclonal), Citrulline (SMC‐5018, Stressmarq Biosciences), Flag (AE169PM, Abclonal), HA (AE105, Abclonal), CD133 (A0219, Abclonal), Nanog (101287‐T32, Sinobiological), His (AE086, Abclonal), USP7 (A3448, Abclonal), IgG (AC005, Abclonal) and CD44 (60224‐1‐Ig, Proteintech).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Knockdown, CCK-8 Assay