Twist1

The immunoblots were processed as described previously [26 (link)]. The primary antibodies included anti-mouse RBMS3 (Sigma-Aldrich, St Louis, MO, USA), β-actin (Cell Signaling Technology, USA), Twist1 (Abcam, Cambridge, MA, USA), anti-rabbit MMP2 (Abcam). The secondary antibodies were purchased from Cell Signaling Technology. The dilutions of antibodies were according to the product usage information.

Total proteins were extracted from the indicated cells with RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA). The protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Western blotting was performed using a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis system. The primary antibodies used for the western blots were as follows: rabbit polyclonal antibodies against EFTUD2 (Novus), vimentin (Cell Signaling Technology), GAPDH (Cell Signaling Technology, 5174), cleaved-caspase 3 (Cell Signaling Technology, 9661), cleaved-caspase 7 (Cell Signaling Technology, 8438), PARP (Cell Signaling Technology, 9532), MCL-1 (Cell Signaling Technology, 94296), and pSTAT3 (Cell Signaling Technology, 9145) and monoclonal antibodies against E-cadherin (BD Biosciences, San Jose, CA, USA, 564186), Twist1 (Abcam, Cambridge, UK, ab50887) and STAT3 (Cell Signaling Technology, 12640).

Human kidney specimens were obtained from diagnostic renal biopsies performed in the Affiliated Hospital of Nanjing University Medical School. Nontumor kidney tissues from the patients who had renal carcinoma and underwent nephrectomy were used as the normal controls. Paraffin-embedded human kidney biopsy sections were prepared using a routine procedure. Kidney biopsy specimens were then stained with Twist1 (Abcam, catalog 50887) antibody.

To prepare whole cell extract, cells were lysed using Pro-prep buffer (Intron Biotechnology, Seoul, Korea) including protease inhibitors. A total of 20–60 μg of protein extract was resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were probed with primary antibodies against CCL7 (#7899F8, Genway, Sandiego, CA, US [7 (link), 14 (link)]A), CCR1 (#3414-100, Biovision, Milpitas, CA, USA), CCR2 (#NB110-55674, Novus, Littleton, USA), CCR3 (#PRS1109, Sigma, St. Louis, MO, USA), CCR5 (#ab32048, Abcam, Cambridge, MA), E-cadherin (#610181, BD Bioscience, San Jose, CA, USA), N-cadherin (#4061, Cell Signaling Technology, Danvers, USA), vimentin (#sc-32322, Santa Cruz Biotechnology, CA, USA), Twist1(#ab50887, Abcam, Cambridge, MA), α-SMA (#SC-53142, Santa Cruz Biotechnology, CA, USA), Snail (#3895, Cell Signaling Technology, Danvers, USA), phospho JNK1/2/3 (#TA312591, Origene, Rockville, MD, USA), total JNK1/2/3 (#TA325661, Origene, Rockville, MD, USA), phospho ERK (#612358, BD Bioscience, San Jose, CA, USA), total ERK (#9102, Cell Signaling Technology, Danvers, USA), and β-actin (#3700, Cell Signaling Technology, Danvers, USA) followed by incubation with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, CA, USA). β-actin was used as a loading control in western blot analysis.

Dihydrorotenone was provided by Dr. Kyeong Kyu Kim at Sungkyunkwan University (Suwon, Korea). An annexin-V staining kit was purchased from BD Bioscience (Franklin Lakes, NJ). An EZ-cytox cell viability assay kit was purchased from Daeill Lab (Seoul, Korea). A cell contraction assay kit was purchased from Cell Biolabs, Inc (San Diego, CA). Twist1 (Cat: ab50887, Abcam, Cambridge, MA), FSP1 (Cat: 07-2274, Millipore, Burlington, MA), PDGFRα (Cat: S3164, Cell Signaling, Danvers, MA), PDGFRβ (Cat: ab32570, Abcam, Cambridge, MA), FAPα (Cat: 53066, Abcam, Cambridge, MA), α-SMA (Cat: (E184) 04-1094, Millipore, Burlington, MA) and α-tubulin antibodies (Cat: SC-8035, Santa Cruz, Dallas, TX) were used for immunoblotting.