Tgfβr1

The generation of p18^−/−, p18^+/−, and p18^−/−:Brca1^+/− mice in Balb/c background, and p18^−/−and p18^−/−;Brca1^f/f; MMTV-Cre (p18^−/−; Brca1^MGKO) mice in Balb/c-B6 mixed background has been previously described [18 (link), 19 (link), 54 (link)]. The Institutional Animal Care and Use Committee at the University of Miami and Shenzhen University approved all animal procedures. Animals were housed in a specific pathogen-free environment with a 12/12 light cycle. Animals were euthanized by exposure to isoflurane followed by cervical dislocation. At least four female mice were analyzed for each genotype, or were transplanted with each type of tumor cells. Histopathology and immunohistochemistry (IHC) were performed as previously described [18 (link), 19 (link), 54 (link)]. The primary antibodies used were: TGFβR1, TGFβR2 (Santa Cruz), p-Smad2, p-FRA1 (Cell signaling), CK5 (Covance), and Vim (Abcam). Immunocomplexes were detected using the Vectastain ABC DAB kit according to the manufacturer’s instructions (Vector Laboratories). The positive results of IHC were quantified by H-score, as previously described [55 (link)].

The proteins suspension of liver tissues were obtained using a total protein extraction kit (APPLYGEN, Beijing, China) and protein concentrations were determined by the BCA protein assay kit while using BSA as a standard. Subsequently, standard western blot procedures were followed, as described elsewhere [41 (link)]. β-actin was used as an endogenous control and blots were quantified by using Image J Software (NIH, Bethesda, MD, USA). The primary and secondary antibodies used for western blot (AMPK, AMPK-p, p-ACC, ACC, CD68, and β-actin) were purchased from Abcam (Cambridge, UK) and other antibodies (CaMKKβ, NF-κβ, TGF-βR1, PPARα, SMAD2/3-P, and SMAD2/3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were lysed in RIPA buffer (Millipore Sigma, St. Louis, MI, USA, R0278) in the presence of protease inhibitors (Roche, 11836170001). Protein lysates were quantified using BCA protein assay (Thermo Scientific, 23225), denatured in 2 × Laemmli sample buffer (Biorad, Hercules, CA, USA, 161-0737) at 95 °C for 10 min, and placed on ice for 2 min. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Millipore, IPVH00010). Membranes were blocked in 5% skim milk in TBS buffer with 0.05% tween-20 (TBS-T), incubated with primary antibody against PD-L1 (Cell Signaling, #13684), p65 (Cell Signaling, #8242), phospho-p65 (Cell Signaling, #3033), phospho-STAT1(Cell Signaling, #9167), STAT1(Cell Signaling, #14994), phospho-STAT3(Cell Signaling, #9145), STAT3(Cell Signaling, #12640), Smad2/3 (Cell Signaling, #3102), phospho-Smad2/3(Cell Signaling, #8828), TGFβR1 (Santa Cruz, sc-518018), or GAPDH (Santa Cruz, Dallas, TX, USA, sc-32233) at 4 °C overnight, washed in TBS-T buffer, and incubated with HRP-conjugated secondary antibody (G.E. Healthcare, Chicago, IL, USA, NA9340) at room temperature for 1 h. Protein bands were visualized using enhanced chemiluminescence (ECL) reaction. The density of protein bands was quantified using Image J software. The density of actin bands was used to normalize the data for protein loading.