Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet:

Article Snippet: SIS3 , MedChemExpress , Cat# HY-13013.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Magnetic Beads, Plasmid Preparation, Extraction, Microarray, Software

Journal: iScience

Article Title: AKT2-mediated nuclear deformation leads to genome instability during epithelial-mesenchymal transition.

doi: 10.1016/j.isci.2023.106992

Figure Lengend Snippet: Figure 4. Smad3 contributes to AKT2 phosphorylation and TGFb-induced nuclear deformation (A) A549 cells were infected with lentiviruses encoding shRNAs specific to luciferase (sh-Luciferase), Smad2 (sh-Smad2), or Smad3 (sh-Smad3). The cells were treated with (+) or without () TGFb for 24 h and lysed. An equal amount of whole cell lysates was analyzed by immunoblotting with anti-Smad2/3 or anti-actin antibody. (B) The cells as described in (A) were treated with or without TGFb for 24 h and stained for lamin A (white) and DNA (blue). Scale bars, 10 mm. The percentage of the cells with a crumpled or lobulated nucleus was measured (n R 600). (C) A549 cells were transiently transfected with the plasmid encoding FLAG-Smad3 or HA-Smad2 for 24 h, and then stained for FLAG (green), HA (green), and DNA (white). Scale bars, 20 mm. The percentage of the transfection-positive cells with a deformed nucleus was measured (n R 269). (D) A549 cells expressing shRNAs to luciferase or Smad3 were treated with or without TGFb for 1 h. An equal amount of whole cell lysates was analyzed by immunoblotting with anti-AKT2 pS474 or anti-AKT2 antibody. The phosphorylation of AKT2 at Ser474 was quantified and expressed as –fold relative to the level of the control. (E) A549 cells were pre-treated with the Smad3 inhibitor SIS3 for 4 h and then co-treated with TGFb for 1 h. An equal amount of whole cell lysates was analyzed by immunoblotting with the antibodies as indicated. The phosphorylation of AKT2 at Ser474 was quantified and expressed as –fold relative to the level of the control. Data information: In B and C, values (means G SD) were from three independent experiments. ***p < 0.001. See also Figure S3.

Article Snippet: SP600125 Merck Millipore Cat#420128 SB23580 Calbiochem Cat#559389 Y27632 EMD Chemicals Cat#688000 Cytochalasin D Sigma-Aldrich Cat#C8273 ML-7 Sigma-Aldrich Cat#12764 Blebbistatin Sigma-Aldrich Cat#B0560 methylstat Sigma-Aldrich Cat#SML0343 SIS3 Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Infection, Luciferase, Western Blot, Staining, Transfection, Plasmid Preparation, Expressing, Control

Journal: Cancers

Article Title: TGF-Beta-Activated Cancer-Associated Fibroblasts Limit Cetuximab Efficacy in Preclinical Models of Head and Neck Cancer

doi: 10.3390/cancers12020339

Figure Lengend Snippet: TGF-beta-activated CAFs reduce sensitivity to cetuximab in vivo, and blocking TGF-beta sensitize HNC-PDX to cetuximab. ( A ) The tumor volume of the CAL33 xerograph model with or without CAFs isolated from PDX #19. 10 6 cells from each cell line (CAL33 tumor cells, PDX #19 CAFs) were injected subcutaneously in NOD/SCID mice. Mice were randomized into 4 arms (tumors, n = 8–16) and treated with cetuximab (10 mg/kg/5d) via intraperitoneal injection or vehicle. The average tumor volumes ± SEM are presented. Statistical significance was calculated by unpaired t -test (* p < 0.05 and *** p < 0.001). ( B ) Tumor weights of the 4 arms. Statistical significance was calculated by unpaired t -test (* p < 0.05 and *** p < 0.001). ( C ) H&E staining. Scale bar: 0.3X, 5000 μm. ( D ) Tumor volume of Cetuximab Prog -PDX (PDX #18). Mice were randomized into 4 arms (tumors, n = 7–8) and treated with vehicle or cetuximab (10 mg/kg/5d) and/or SMAD3 inhibitor SIS3 (5 mg/kg/d) via intraperitoneal injection. The average tumor volumes ± SEM are presented. Statistical significance was calculated by unpaired t -test (* p < 0.05, *** p < 0.001, and **** p < 0.0001). ( E ) Tumor weights of the 4 arms. Statistical significance was calculated by unpaired t -test (* p < 0.05 and *** p < 0.001). ( F ) H&E staining. Scale bar: 0.3X, 5000 μm.

Article Snippet: The SMAD3 inhibitor, SIS3 (APExBIO, Houston, TX, USA), was dissolved in 0.5% DMSO and then with corn oil for a final working concentration of 5 mg/kg.

Techniques: In Vivo, Blocking Assay, Isolation, Injection, Staining

Journal: Cancers

Article Title: TGF-β Signaling Loop in Pancreatic Ductal Adenocarcinoma Activates Fibroblasts and Increases Tumor Cell Aggressiveness

doi: 10.3390/cancers16213705

Figure Lengend Snippet: TGF-β released by tumor cells promotes fibroblasts’ activation. ( A ) Expression of α-SMA in MRC-5 fibroblasts, cultured on the upper side of the transwell, at the end of 5 days exposure to PANC-1 cells, or TGF-β (10 ng/mL); representative cropped images of the Western blot of α-SMA and GAPDH (housekeeping protein) derived from the original blots in . ( B ) The intensity of the SMA signal was quantified and normalized to that of GAPDH from the same lane (α-SMA expression in MRC-5 cells in the monoculture was set to 1. Mean values ± SE (n = 3 biological replicates) were compared by one-way ANOVA; * p < 0.05. ( C ) MRC-5 fibroblasts at day 5 of the monoculture or treated with TGF-β (5 ng/mL), with or without 10 μM TGF-β receptor inhibitor (SB431542) or SMAD3 inhibitor (SIS3), or day 5 of the co-culture with PANC-1 cells, in the absence or presence of the same inhibitors. The number of MRC-5 cells in the monoculture was set as a reference at 100%. Data, represented as box-and-whisker plots, were compared by one-way ANOVA; **** p < 0.0005, *** p < 0.001, ** p < 0.01 (n = 3–13).

Article Snippet: The TGF-β receptor 1 (TGF-βR1) inhibitor SB431542 (S1067—10 mg, Selleck, by Aurogene, Rome, Italy) and the SMAD3 inhibitor SIS3 (A16065—5 mg, Adooq Bioscience, by CliniSciences, Rome, Italy) were kept at −80 °C and diluted in sterile 100% ethanol solvent.

Techniques: Activation Assay, Expressing, Cell Culture, Western Blot, Derivative Assay, Co-Culture Assay, Whisker Assay

Journal: Cancers

Article Title: TGF-β Signaling Loop in Pancreatic Ductal Adenocarcinoma Activates Fibroblasts and Increases Tumor Cell Aggressiveness

doi: 10.3390/cancers16213705

Figure Lengend Snippet: ( A ) MRC-5 cells promote tumor-cell proliferation in transwell co-cultures. PANC-1 cells in the lower chamber of the transwell units were counted after 5 days of co-culture with MRC-5 cells or exposure to TGF-β (5 ng/mL), with or without 10 μM TGF-β receptor inhibitor (SB431542) or SMAD3 inhibitor (SIS3). The number of PANC-1 cells in the monoculture was set as a reference at 100%. ( B ) MRC-5 cells promote tumor-cell chemoresistance to gemcitabine (Gem). The viability of PANC-1 cultured for 5 days with or without MRC-5 fibroblasts in the transwell, or with or without TGF-β (5 ng/mL), and then treated with 50 µM Gem for 48 h, was assessed by MTT. The viability of the untreated monoculture was set to 100%. Data, represented as box-and-whisker plots, were compared by one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0005 (( A ): n = 4–10; ( B ): n = 4).

Article Snippet: The TGF-β receptor 1 (TGF-βR1) inhibitor SB431542 (S1067—10 mg, Selleck, by Aurogene, Rome, Italy) and the SMAD3 inhibitor SIS3 (A16065—5 mg, Adooq Bioscience, by CliniSciences, Rome, Italy) were kept at −80 °C and diluted in sterile 100% ethanol solvent.

Techniques: Co-Culture Assay, Cell Culture, Whisker Assay

Journal: Cancers

Article Title: TGF-β Signaling Loop in Pancreatic Ductal Adenocarcinoma Activates Fibroblasts and Increases Tumor Cell Aggressiveness

doi: 10.3390/cancers16213705

Figure Lengend Snippet: Role of TGF-β in PANC-1/MRC-5 heterospheroid proliferation. ( A ) Representative images of heterospheroids consisting of PANC-1 and MRC-5 cells pre-labeled with Vybrant ® CFDA SE and CellTracker™ Deep Red dye. Scale bars: 200 µm. ( B ) Images of MRC-5/PANC-1 heterospheroids treated with 10 μM TGF-βR1 inhibitor (SB431542) or SMAD3 inhibitor (SIS3) for 7 days. ( C ) The size of the spheroids was determined using the acquired images (average of 4 different diameters for each condition). Data, represented as box-and-whisker plots, were compared by one-way ANOVA. * p < 0.05 (n = 10–12).

Article Snippet: The TGF-β receptor 1 (TGF-βR1) inhibitor SB431542 (S1067—10 mg, Selleck, by Aurogene, Rome, Italy) and the SMAD3 inhibitor SIS3 (A16065—5 mg, Adooq Bioscience, by CliniSciences, Rome, Italy) were kept at −80 °C and diluted in sterile 100% ethanol solvent.

Techniques: Labeling, Whisker Assay