Am17100 pm10050

HTR-8/SVneo cells were a generous gift from Charles Graham (Queen's University, Kingston, Canada) and were cultured in RPMI1640 (Gibco, 11965-092) supplemented with 10% fetal bovine serum, heat inactivated, 1% penicillin/streptomycin, and 1% L-glutamine. Antibodies for TβR3 (Abcam, ab166705; used at a dilution of 1/500), Par6 (Abcam, ab180159; used at a dilution of 1/1000), TUBB (Abcam, ab6046; used at a dilution of 1/10000), ACTB (Cell Signaling, 4967; used at a dilution of 1/10000), RhoA (Cell Signaling, 2117; used at a dilution of 1/1000), SMAD2/3 (Cell Signaling, 3102; used at a dilution of 1/1000), and phosphorylated SMAD2/3 (Cell Signaling, 3101; used at a dilution of 1/1000) were purchased. siH19 (Ambion, 4390816/n272452), siTβR3 (Dharmacon, L-010545-00), siPar6 (Ambion, 4392420/s27161), siCon (Ambion, Am4636), iLet7 (Ambion, 4392431), iCon (Ambion, AM17010), let-7 mimics (let-7) (Ambion, AM17100/PM10050) and miCon (Ambion, AM17110) were purchased. Plasmids expressing human H19 (pH19) and empty vector were previously described [14 (link), 16 (link)]. TGF-β2 cytokine was purchased (Cell Signaling, 8406).

Antibodies for SAHH (for RNA immunoprecipitation, Santa Cruz, Dallas, TX, USA; sc-271389; for western blot, Proteintech Group, Chicago, IL, USA; 10757-2-P), DNMT3B (Novus, Littleton, CO, USA; NB300-516), KSRP (Cell Signaling, Boston, MA, USA; 13398), Phospho-AMPK-alpha (Thr172) (Cell Signaling; 2535), AMPK alpha (Cell Signaling; 2532), β-tubulin (Abcam, Cambridge, MA, USA; ab6046), β-actin (Abcam; ab8226) and mouse preimmune IgGs (Chemicon, Billerica, MA, USA; PP54) were purchased. Control siRNA (siCon; Ambion, Grand Island, NY, USA; AM4636), siRNAs specific for human H19 (siH19; Ambion; 4390771/n272452), KSRP (siKsrp; Ambion; 4390824/s16322) and DNMT3B (siDNMT3B; Santa Cruz; SC-37759), and let-7a mimics (Let-7; Ambion; AM17100/PM10050), Pre-miR negative control (miCon; Ambion; AM17110), Let-7 inhibitor (iLet-7; Ambion; 4392431) and miRNA control (Ambion; AM17010) were purchased. Metformin (ALX-270-432-G005), AICAR (123040) and D-Eritadenine (sc-207632) were from ENZO Life Sciences International Inc. (Uniondale, NY, USA) Calbiochem (Billerica, MA, USA), and Santa Cruz, respectively. Metformin and AICAR were used at final concentrations of 2 and 0.5 mm, respectively.