Ecl detection reagent

The cellular proteins were extracted and analyzed for protein expression as previously described [12 (link)]. Briefly, thirty micrograms of the cellular proteins were resolved by electrophoresis in 10 % SDS-polyacrylamide gel, and subsequently transferred to polyvinylidene difluoride (PVDF) membrane. Following 1 h incubation in a fresh TBS buffer containing 0.1 % Tween-20 and 5 % BSA, the blots were probed with specific antibodies including anti-p-ERK, anti-p-p38 and anti-β-actin. The bound primary antibodies were detected by HRP conjugated anti-mouse IgG accordingly. The activity of peroxidase on the blot was visualized by enhanced chemiluminescence (ECL) detection reagents (GE Healthcare, Sweden).

The cellular proteins were extracted and analyzed for protein expression as previously described [14 (link)]. Briefly, thirty micrograms of the cellular proteins were resolved by electrophoresis in 10% SDS-polyacrylamide gel and subsequently transferred to polyvinylidene difluoride (PVDF) membrane. Following 1 h incubation in a fresh TBS buffer containing 0.1% Tween-20 and 5% BSA, the blots were probed with specific antibodies including anti-HO-1, anti-NQO1, anti-β-actin, anti-phospho-Akt, anti-Nrf2, or anti-lamin b1. The bound primary antibodies were detected by horseradish peroxidase conjugated anti-rabbit IgG or anti-goat IgG accordingly. The activity of peroxidase in the blot was visualized by enhanced chemiluminescence (ECL) detection reagents (GE Healthcare, Sweden).

For a full list of antibodies, see reporting summary. The samples were prepared in NuPAGE Sample Buffer (Invitrogen). Then proteins were separated by electrophoresis in 4–12% Bis-Tris Protein Gels and transferred to the polyvinylidene difluoride membrane (Thermo Scientific). The membranes were blocked in 5% dry milk in 0.1% Tween-20 in PBS and detected with the indicated antibodies. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (BIO-RAD), proteins were visualized using ECL detection reagents (GE Healthcare). Uncropped gel images for Western blots are available in Source Data file.

Western blots were either performed from total cell lysates obtained by lysing cells directly with RIPA buffer (Sigma-Aldrich) complemented with protease inhibitor cocktail (cOmplete, Roche) or from fractionated lysates (see below). Proteins concentration was determined using BCA protein assay kit (Thermo). SDS-PAGE electrophoresis was carried out in 10% pre-cast polyacrylamide gels (Novex) in MOPS buffer. After transfer onto nitrocellulose, membranes were blocked for 1hr with 10% non-fat milk in TBS-0.1% Tween solution and probed overnight with primary antibody: Ascl1 (1:300, hybridoma supernatant, gift from David Anderson and François Guillemot), Histone H3 (1:10,000, Abcam, ab1791) and α-Tubulin (1:10,000, Abcam, ab7291), LaminB (1:500, Santa Cruz, sc-6216) and Huwe1 (1:1,000, Bethyl Laboratories, A300-486A). Mouse secondary (goat anti-mouse HRP 1:10,000, Millipore, 71045-3) or rabbit secondary (1:5,000, Rabbit IgG HRP linked, GE Healthcare, NA934V) antibodies were used for detection in 5% milk in TBS-0.1% Tween solution for 1 hour. Membranes were developed using ECL Prime (GE Healthcare, RPN2232) for Ascl1 or ECL detection reagents (GE Healthcare, RPN2106) for the other antibodies. All western blots were performed at least in triplicate.

Cells were washed twice with cold PBS and lysed in the 2x LSB buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl pH 6.8). Cell extracts were separated by SDS-PAGE and transferred to the nitrocellulose or PVDF membranes (GE Healthcare). The membranes were blocked in 5% dry milk in 0.1% Tween-20 in PBS and probed with primary antibodies. After incubation with HRP-conjugated secondary antibodies (Vector Laboratories and Santa Cruz Biotechnology), proteins were visualized using ECL detection reagents (GE Healthcare). The primary antibodies used: phospho-ATM (S1981) (1:500, ab81292, Abcam), phospho-Chk2 (Thr68) (1:200, 2661, Cell Signalling), phospho-ATR (Ser428) (1:200, 2853, Cell Signalling), phospho-Chk1 (Ser345) (1:300, 2348, Cell Signalling), phospho-RPA32 (S33) (1:1000, A300-246A, Bethyl), gamma H2A.X (phospho-S140) (1:2000, ab22551, Abcam), H2AX (1:5000, NB100-638, Novus Biologicals), phospho-Rb (Ser807/811) (1:1000, 9308, Cell Signalling), p53 (DO-1) (1:1000, sc-126, Santa Cruz), p21 (H-164) (1:400, sc-756, Santa Cruz), phospho-p70 S6 Kinase (Thr389) (1:1000, 9206, Cell Signalling), p62 (1:1000, GP62-C, PROGEN Biotechnik), LC3B (D11) XP™ (1:1000, 3868, Cell Signalling), β-actin (1:10.000, A1978, Sigma-Aldrich), ATG5 (C-terminal) (1:700, A0731, Sigma-Aldrich), ATG7 (D12B11) (1:1000, 8558S, Cell Signalling).

TGFβ1 was purchased from Thermo Fisher Scientific, Waltham, MA, USA. Cells were lysed in RIPA lysis buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, 10 mM NaF, 0.1% SDS, 1% Nonidet P-40, 1 × protease inhibitor mixture (Roche, Penzberg, Germany). Blots were performed for E-cadherin, β-catenin, Vimentin (BD Biosciences, San Jose, CA, USA), ZEB1, Snail (Abcam, Cambridge, MA, USA), phospho-Smad2/3 (Ser465/467) (Cell Signaling Technology, Danvers, MA, USA), Shc1 (BD Biosciences) or horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), followed by ECL detection reagents (GE Healthcare, Pittsburgh, PA, USA). Blots were stripped and reprobed for total Smad2/3 (Cell Signaling Technology) or β-actin (Chemicon, Darmstadt, Germany).

Western blotting was performed as described previously [13 (link)]. Briefly, cytoplasmic protein fractions of SMMC-7721 or Bel-7402 spheres cells were prepared using a NucBuster Protein Extraction kit (Novagen, Merck, Darmstadt, Germany). After protein quantification by the method of Bensadoun and Weinstein [14 (link)], cytoplasmic proteins were separated by 10% sodium dodecyl sulfate (SDS) − polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After sequential incubation of the membranes with a primary antibody and a horseradish peroxidase-conjugated secondary antibody, the immunoblots were visualized using enhanced chemiluminescence (ECL) detection reagents (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Quantification of the detected bands was carried out, and the ratio of phosphorylated protein bands to total protein bands is shown in the figures. Since some studies have shown that phosphorylation of Akt at T308 is a more valuable biomarker for tumor progression than phosphorylation of Akt at S473, we used anti-p-Akt (T308) for Western blot analysis of Akt phosphorylation [15 (link), 16 (link)].