Journal: bioRxiv

Article Title: Subjugation of TGFβ Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End-Joining

doi: 10.1101/353441

Figure Lengend Snippet: (A) TCGA tumor sample clustering of 243 HPV− and 36 HPV+ HNSCC using a gene-set signature upregulated by TGFβ signaling. (B) Percentage of p-SMAD2 positive cells after treatment with TGFβ or TβRI inhibitor LY364947 for a representative HPV− cell line, SAS (black), and a HPV+ cell line, SCC47 (red). Two-tailed Student’s t-test; **, P < 0.01; **, P < 0.005. (C) The percentage of p-SMAD2 positive cells following TGFβ treatment of HPV− cell lines (black) and HPV+ cell lines (red). (D) Data from panel C grouped according to their HPV status. (E) Representative images of p-SMAD2 immunostaining of HNSCC PDX (left panel). The percentage of p-SMAD2 positive cells induced by TGFβ in HPV− (black) and HPV+ (red) PDX (right panel). (F) Data from panel E grouped according to HPV status. (G) Distribution of p-SMAD2 scores as described in methods for 130 HPV− and 65 HPV+ HNSCC. Panel D, F and G, Mann-Whitney U test; *, P < 0.05; ***, P < 0.005.

Article Snippet: Cells were incubated with γH2AX mouse monoclonal antibody (EMD Millipore) at 1:500 dilution, RAD51 rabbit polyclonal antibody (Santa Cruz) at 1:200, Geminin rabbit polyclonal antibody (Abcam) at 1:200, or phospho-SMAD2 on serine 465/467 at 1:200 (Cell Signaling) at room temperature for 2 hr or 4°C overnight.

Techniques: Two Tailed Test, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Subjugation of TGFβ Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End-Joining

doi: 10.1101/353441

Figure Lengend Snippet: (A) Number of γH2AX foci per irradiated (2 Gy) HPV− cell lines (white) and HPV+ cell lines (red) at 30 min post irradiation pre-treated with (hatched) or without (open) TβRI inhibitor LY364947. (B) Number of γH2AX foci per cell in irradiated (2 Gy) HPV− (white) and HPV+ (red) PDX pre-treated with (hatched) or without (open) LY364947. (C) Correlation of percent nuclear p-SMAD2 positive cells following TGFβ treatment (from and E) with γH2AX foci (from panel A and B) of HPV+ (red) and negative (black) cell lines (triangles) and PDX (squares). Linear regression analysis was used to calculate P value and R 2 . (D) Number of γH2AX foci per cell of irradiated (2 Gy) SAS cells treated with inhibitors against ATM (ATMi, KU55933), DNA-PK (DNA-PKi, KU57788), or ATR (ATRi, AZD6738) with or without LY364947. Two-tailed Student’s t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Article Snippet: Cells were incubated with γH2AX mouse monoclonal antibody (EMD Millipore) at 1:500 dilution, RAD51 rabbit polyclonal antibody (Santa Cruz) at 1:200, Geminin rabbit polyclonal antibody (Abcam) at 1:200, or phospho-SMAD2 on serine 465/467 at 1:200 (Cell Signaling) at room temperature for 2 hr or 4°C overnight.

Techniques: Irradiation, Two Tailed Test

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Si-Miao-Yong-An decoction attenuates cardiac fibrosis via suppressing TGF-β1 pathway and interfering with MMP-TIMPs expression.

doi: 10.1016/j.biopha.2020.110132

Figure Lengend Snippet: Fig. 4. SMYAD improved cardiac fibrosis by inhibiting TGF-β1/Smad pathway (A) Representative immunoblots of protein in TGFβ1/Smad pathway. Western-blot analysis of (B) TGF-β1, (C) p-Smad2, (D) p-Smad3, (E) Smad4, (F) Smad7. Sham (n=6), TAC (n=6), TAC + Captopril (n=6), TAC + SMYAD (n=6). ### P < 0.001, ## P < 0.01, # P < 0.05 vs the Sham group; *** P < 0.001, ** P < 0.01, * P < 0.05 vs the TAC group.

Article Snippet: After blocking with 5 % non-fat milk for 1 h at room temperature, the blots were incubated overnight at 4 °C with the following antibodies: Col1 (Collagen Type I, 1:1000, Proteintech Cat 14695-1-AP); Col3 (Collagen Type III (N-Terminal), 1:1000, Proteintech Cat 22734-1-AP); αSMA (α-Smooth Muscle Actin, 1:1000, Cell Signaling Technology Cat 19245); MMP1 (MMP1, 1:1000, Proteintech Cat 10371-2-AP); MMP9 (MMP9 (N-Terminal), 1:1000, Proteintech Cat 10375-2-AP); TIMP1 (TIMP1, 1:1000, ab179580, Abcam); TIMP2 (TIMP2, 1:1000, ab180630, Abcam); CTGF (CTGF, 1:1000, Proteintech Cat 23936-1-AP); TGFβ1, (TGFβ1, 1:1000, Cell Signaling Technology Cat 3711); p-SMAD2 (Phospho-SMAD2, 1:500, Cell Signaling Technology Cat 18338); t-SMAD2 (SMAD2, 1:1000, Proteintech Cat 12570-1-AP); p-SMAD3 (Phospho-SMAD3, 1:500, Cell Signaling Technology Cat 9520); t-SMAD3 (SMAD3, 1:1000, Proteintech Cat 25494-1-AP); SMAD4 (SMAD4, 1:1000, Proteintech Cat 10231-1-AP); SMAD7 (SMAD7, 1:1000, Proteintech Cat 25840-1-AP); p-TAK1 (Phospho-TAK1, 1:1000, Cell Signaling Technology Cat 9339); t-TAK1 (TAK1, 1:1000, Cell Signaling Technology Cat 5206); p-p38 (Phospho-p38 MAPK, 1:1000, Cell Signaling Technology Cat 4511); t-p38 (P38 MAPK, 1:1000, Proteintech Cat 14064-1-AP); GAPDH (1:1,000, Cell Signaling Technology Cat 2118) was used as protein loading controls for shown in the figures.

Techniques: Western Blot

Journal: BMC cancer

Article Title: A novel TGF-β receptor II mutation (I227T/N236D) promotes aggressive phenotype of oral squamous cell carcinoma via enhanced EGFR signaling.

doi: 10.1186/s12885-020-07669-5

Figure Lengend Snippet: Fig. 1 Effect of I227T/N236D TβRII mutation on TGF-β signaling. a. Transcriptional activities were assessed via luciferase assay after transfection with pcDNA3 vector (cont), wild-type (WT), I227T (227), N236D (236), and I227T/N236D (227/236) mutant TβRII constructs. DR26 cells were co-transfected with p3TP-lux promoter-reporter construct, pRL-TK, and TβRII constructs. Transfected cells were incubated for 24 h in DMEM supplemented with 0.2% FBS in the presence of vehicle (TGF-β (−)) or 1 ng/ml TGF-β1 (TGF-β (+)). Transfection efficiency was normalized using Renilla luciferase. Data represent mean ± standard deviation (*P < 0.05). b. Stable transfectant cells were constructed by transfection of pIRES2-EGFP vector (IRES), wild-type TβRII (WT), and I227T/N236D TβRII (227/236) constructs into HSC-2 cells. TβRII expression in stable cells was confirmed by western blotting. Protein samples of IRES, WT, and 227/236 were separated on the same gel and the protein bands were cropped. Uncropped blots were shown in Additional file 1: Figure S1. c. Stable HSC-2 cells harboring empty vector (IRES), wild-type TβRII (WT), and I227T/N236D TβRII (227/236) were mock-treated or treated with 10 ng/ml TGF-β1 for 18 h. Smad2 protein level (t-Smad2) and the phosphorylation level of Smad2 (p-Smad2) were determined by western blotting. Data represent mean ± standard deviation from three independent experiments (*P < 0.05, **P < 0.01). Full length immunoblots were shown in Additional file 2: Figure S2

Article Snippet: Antibodies against phospho-Smad2 (Ser465/467, #3101, 1: 1000), Smad2 (#3122, 1:1000), cleaved Caspase-3 (#9661, 1:1000), cleaved PARP (#9541, 1:1000), epidermal growth factor receptor (EGFR) (#2232, 1:1000), phospho-EGFR (Tyr1068, #2234, 1:1000), phospho-Akt (Ser473, #9271, 1: 1000), Akt (#9272, 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Construct, Incubation, Standard Deviation, Expressing, Western Blot, Phospho-proteomics