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) is highly expressed in oesophageal squamous cell carcinoma (OSCC) tissues and linked to tumour stage and poor prognosis. (A) Volcano plot showing gene expression differences between cisplatin‐resistant and parental groups in GSE169337. (B) Kaplan–Meier survival analysis of OSCC patients with different PADI4 levels using The Cancer Genome Atlas (TCGA) data. (C) Immunohistochemical (IHC) analysis comparing PADI4 expression in OSCC tissues to normal oesophageal tissues. (D) IHC analysis of PADI4 expression across TNM stages in OSCC. (E, F) Kaplan–Meier analysis of overall and recurrence‐free survival in OSCC patients with varying PADI4 expression from IHC data. (G) PADI4 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (H) PADI4 knockdown markedly inhibited the migration ability of ECA109 and KYSE150 cells indicated by transwell assay.

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: Gene Expression, Immunohistochemical staining, Expressing, Knockdown, Colony Assay, Migration, Transwell 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: Clinical characteristics of patients and expression of peptidylarginine deiminase 4 (PADI4).

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

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: Regression analysis of oesophageal squamous cell carcinoma (OSCC) patients.

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:

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: Cancer stem cell (CSC)‐like phenotypes and cisplatin resistance of oesophageal squamous cell carcinoma (OSCC) cells were suppressed by peptidylarginine deiminase 4 (PADI4) knockdown. (A) Comprehensive analysis of PADIs and the stemness across 33 tumour types within the The Cancer Genome Atlas (TCGA) database. (B) Correlation between PADI4 and stemness markers (CD133, LGR5, Nanog, OCT4 and CD24) in GSE23400. (C, D) qPCR showed PADI4 knockdown significantly reduced CD133 and Nanog mRNA levels in ECA109 (C) and KYSE150 (D) cells. (E, F) Western blot revealed PADI4 knockdown significantly decreased CD133 and Nanog protein levels in ECA109 (E) and KYSE150 (F) cells. (G, H) Western blot showed PADI4 plasmids significantly increased CD133 and Nanog protein levels in ECA109 (G) and KYSE150 (H) cells. (I) PADI4 knockdown significantly reduced sphere formation in ECA109 and KYSE150 cells. (J, K) Downregulation of PADI4 enhanced the resistance of ECA109 (J) and KYSE150 (K) cells to cisplatin through CCK8 assay. (L, M) Limiting dilution assays showing the self‐renewing capacity of PADI4 downregulated in CD133‐positive cells of ECA109 and KYSE150.

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: Knockdown, Western Blot, 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: 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: 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: Peptidylarginine deiminase 4 (PADI4) facilitates tumour growth and metastasis in vivo, while GSK484 exhibits a synergistic effect with cisplatin in vivo. (A) A schematic diagram illustrating the treatment protocol for mice in this study is presented. (B, C) Images of excised subcutaneous tumours from mice treated with shPADI4, GSK484 and cisplatin, and the combination of GSK484 and cisplatin are shown. (D) Immunohistochemical (IHC) analysis of PADI4 protein expression in excised tumours from the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is presented. (E) The volume of subcutaneous tumours in the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is depicted. (F) The weight of subcutaneous tumours in the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is reported. (G) HE staining revealed oesophageal squamous cell carcinoma (OSCC) metastases in the lung tissues. (H) The schematic diagram of the underlying mechanism in this study was shown.

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: In Vivo, Immunohistochemical staining, Expressing, Staining

Journal: BMC Cancer

Article Title: Overexpression of KRAS enhanced the stemness of esophageal cancer cells inhibited by overexpression of circ0043898

doi: 10.1186/s12885-025-14358-8

Figure Lengend Snippet: RNA sequencing reveals gene expression changes and functional enrichment following circ0043898 overexpression in EC cells. ECA109 and Kyse-520 cells were transfected with empty PCDH plasmid (named the PCDH-NC group) and PCDH-circ0043898 plasmid (named the PCDH-circ0043898 group), respectively. Samples were collected for RNA sequencing 24 h post-transfection. ( A ) Venn diagram showing DEGs in Kyse-520 and ECA109 cells following circ0043898 overexpression. ( B ) Heatmap of DEGs in Kyse-520 and ECA109 cells. ( C ) Top 15 GO functional enrichment analysis of 309 commonly DEGs in Kyse-520 and ECA109 cells. ( D ) Top 15 KEGG pathway enrichment analysis of 309 commonly DEGs. ( E ) qRT-PCR detection of KRAS and PI3K mRNA levels in EC cells following circ0043898 overexpression. ( F ) Western blot analysis of KRAS, PI3K, and p-PI3K protein expression in EC cells following circ0043898 overexpression. Blots were cut prior to antibody incubation. *** P < 0.001

Article Snippet: Membranes were blocked with 5% BSA for 1 h, followed by overnight incubation at 4 °C with primary antibodies specific to KRAS (Proteintech, cat 12063-1-AP), PI3K (ABclonal, cat A18355), phospho-PI3K (ABclonal, cat AP0854), CD44 (ABclonal, cat A19020), and CD133 (ABclonal, cat A0219).

Techniques: RNA Sequencing, Gene Expression, Functional Assay, Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing, Incubation

Journal: BMC Cancer

Article Title: Overexpression of KRAS enhanced the stemness of esophageal cancer cells inhibited by overexpression of circ0043898

doi: 10.1186/s12885-025-14358-8

Figure Lengend Snippet: Overexpression of KRAS reversed the stemness inhibition induced by circ0043898 in EC cells. ECA109 and Kyse-520 cells were transfected with PCDH-NC (PCDH-NC group), PCDH-circ0043898 (PCDH-circ0043898 group), or co-transfected with PCDH-circ0043898 and PCDH-KRAS (PCDH-circ0043898 + KRAS group). After 24 h of transfection, samples were collected for qRT-PCR analysis and stem cell spheroid formation assays. After 48 h of transfection, IF and flow cytometry were performed to assess stemness markers CD44 and CD133. ( A ) qRT-PCR analysis of circ0043898, KRAS, and PI3K mRNA expression. ( B ) IF detection of CD44 and CD133 protein expression. ( C ) Flow cytometry analysis of CD44⁺CD133⁺ double-positive cell ratios. ( D ) qRT-PCR analysis confirming KRAS overexpression. ( E ) Western blot detection of KRAS expression in each group. Blots were cut prior to antibody incubation. ( F ) Representative images of stem cell spheroid formation in each group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Membranes were blocked with 5% BSA for 1 h, followed by overnight incubation at 4 °C with primary antibodies specific to KRAS (Proteintech, cat 12063-1-AP), PI3K (ABclonal, cat A18355), phospho-PI3K (ABclonal, cat AP0854), CD44 (ABclonal, cat A19020), and CD133 (ABclonal, cat A0219).

Techniques: Over Expression, Inhibition, Transfection, Quantitative RT-PCR, Flow Cytometry, Expressing, Western Blot, Incubation

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. 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: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT3 inhibitors on the growth and viability of BCa J82, NBT-II, and MB49 cells. Chemical structure of STAT3 inhibitors ( A ) GLG-302, ( B ) TTI-101, and ( C ) SH5-07. ( D ) Basal expression levels of STAT3 and pSTAT3 in BCa cell line (NBT-II, J82, and MB49). ( E – J ) The MTT assay was used to determine the viability of BCa cells at 24 h and 48 h, as shown in graphs. ( K ) IC50 values of compounds on NBT-II, J82, and MB49 cells at 48 h of treatment. Cells were cultured with various doses of STAT3 inhibitors (1–50 µM) and processed for the MTT experiment, as detailed in the Materials and Methods Section. BCa cells were treated with different concentrations of STAT3 inhibitors (0, 6, 12.5, 25, and 50 µM) for 48 h and live cells were counted using the trypan blue dye exclusion method ( L – N ). The results are representative of three independent experiments. Error Bars = Standard error of mean (SEM); * p < 0.05; ** p < 0.001; *** p < 0.0001.

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Expressing, MTT Assay, Cell Culture

Journal: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT3 inhibitors on proliferation of BCa spheroids. Representative images of spheroids generated using J82 ( A ), NBT-II ( B ), and MB49 ( C ) BCa cell lines. All images were captured at a 10X magnification. Growth curves of spheroid sizes ( D ). Schematic diagram illustrating treatment of spheroids with STAT3 inhibitors ( E ). Intracellular ATP content in ( I – K ) spheroids measured by luminescence on day six ( F – H ). Treatment with STAT3 inhibitors decreased the BCa spheroids size; bar represents 200 µM diameter ( I – K ). Spheroids were stained with calcein AM for live cells (green) and EtBr for dead cells (red). Representative images of control vs. treated BCa spheroids ( L , M ). All experiments were performed in triplicate ( n = 3). Values are expressed as the mean ± SEM. Significance is indicated by * p < 0.05, ** p < 0.001, and *** p < 0.0001.

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Generated, Staining, Control

Journal: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT3 inhibitors on apoptosis induction in BCa spheroids. BCa spheroids were treated with STAT3 inhibitors, stained with MitoSOX, and visualized under a fluorescent microscope ( A ). Spheroids stained with MitoTracker (red) and CellEvent Caspase-3/7 (green). Images were acquired using 50X objectives ( B , C ). Protein lysate was prepared from control (CON) and STAT3 inhibitor-treated BCa spheroids (TTI-101- or SH5-07(12.5 µM)) for 6 days to determine the expression of STAT3, pSTAT3, Cyclin D1, PCNA, Caspase 3, cleaved PARP, CD133, and CD44. β-actin was used as a loading control ( D ). The intensity of the indicated protein expression in J82 spheroids ( E ) and NBT-II spheroids ( F ) was quantified using ImageJ software. Data represent the average of three independent experiments and are reported as the mean ± SEM (* p < 0.05, ** p < 0.001, *** p < 0.0001).

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Staining, Microscopy, Control, Expressing, Software

Journal: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT3 inhibitors on BBN-induced rat tumor-derived BCa organoids. Schematic of organoid culture procedure from rat bladder tumors ( A ). Overview of the timeframe experiment ( B ). STAT3 inhibitors (TTI-101 and SH5-07) decreased organoid growth ( C ). Tumor cell proliferation in control (CON) and STAT3 inhibitor-treated organoids was measured using the CellTiter-Glo assay after six days of drug treatment ( D ). Assessment of STAT3 inhibitors on bladder organoids using ethidium homodimer (dead) and calcein AM (live) staining ( E ). Values are ( n = 3) mean ± SEM (** p < 0.001, *** p < 0.0001).

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Derivative Assay, Control, Glo Assay, Staining

Journal: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT 3 inhibitors on apoptosis induction in BCa organoids. Fluorescence staining of mitochondrial superoxide production after treatment with TTI-101 and SH5-07 at concentration of 12.5 µM for six days ( A ). After TTI-101 and SH5-07 treatment, organoids were stained with MitoTracker (red) and CellEvent Caspase-3/7 (green) to detect cell death within organoids ( B ). Protein lysate was prepared from control (CON) and STAT3 inhibitor-treated BBN-rat BCa organoids (TTI-101 or SH5-07 (12.5 µM)). STAT3, pSTAT3, Cyclind1, PCNA, caspase 3, cleaved PARP, and CD133 and CD44 protein expression levels were detected by immunoblotting. β-actin was used as a loading control ( C ). Densitometric analysis was performed to quantify the protein expression in the BCa organoids ( D ). Values are ( n = 3) mean ± SEM (* p < 0.05, ** p < 0.001, *** p < 0.0001). Graphical summary of the molecular effects of the STAT3 inhibitors on BCa cells ( E ).

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Fluorescence, Staining, Concentration Assay, Control, Expressing, Western Blot

Journal: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT3 inhibitors on apoptosis induction in BCa spheroids. BCa spheroids were treated with STAT3 inhibitors, stained with MitoSOX, and visualized under a fluorescent microscope ( A ). Spheroids stained with MitoTracker (red) and CellEvent Caspase-3/7 (green). Images were acquired using 50X objectives ( B , C ). Protein lysate was prepared from control (CON) and STAT3 inhibitor-treated BCa spheroids (TTI-101- or SH5-07(12.5 µM)) for 6 days to determine the expression of STAT3, pSTAT3, Cyclin D1, PCNA, Caspase 3, cleaved PARP, CD133, and CD44. β-actin was used as a loading control ( D ). The intensity of the indicated protein expression in J82 spheroids ( E ) and NBT-II spheroids ( F ) was quantified using ImageJ software. Data represent the average of three independent experiments and are reported as the mean ± SEM (* p < 0.05, ** p < 0.001, *** p < 0.0001).

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Staining, Microscopy, Control, Expressing, Software

Journal: Cells

Article Title: Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

doi: 10.3390/cells13171463

Figure Lengend Snippet: Effect of STAT 3 inhibitors on apoptosis induction in BCa organoids. Fluorescence staining of mitochondrial superoxide production after treatment with TTI-101 and SH5-07 at concentration of 12.5 µM for six days ( A ). After TTI-101 and SH5-07 treatment, organoids were stained with MitoTracker (red) and CellEvent Caspase-3/7 (green) to detect cell death within organoids ( B ). Protein lysate was prepared from control (CON) and STAT3 inhibitor-treated BBN-rat BCa organoids (TTI-101 or SH5-07 (12.5 µM)). STAT3, pSTAT3, Cyclind1, PCNA, caspase 3, cleaved PARP, and CD133 and CD44 protein expression levels were detected by immunoblotting. β-actin was used as a loading control ( C ). Densitometric analysis was performed to quantify the protein expression in the BCa organoids ( D ). Values are ( n = 3) mean ± SEM (* p < 0.05, ** p < 0.001, *** p < 0.0001). Graphical summary of the molecular effects of the STAT3 inhibitors on BCa cells ( E ).

Article Snippet: The following primary antibodies were used: STAT3 (CST-30835, 1:1000), p-STAT3 (CST-9145, 1:1000), Cyclin D1(CST-55506, 1:1000), proliferating cell nuclear antigen (PCNA; CST-13110, 1:1000), caspase 3 (CST-9662, 1:1000), cleaved PARP (CST-5625, 1:1000), CD44 (CST-37259, 1:1000), and CD133 (AB-A0219, 1:1000) and Rabbit secondary antibody (CST-7074, 1:10000).

Techniques: Fluorescence, Staining, Concentration Assay, Control, Expressing, Western Blot