Ab32087

Cells were fixed with 4% paraformaldehyde/PBS for 15 min and permeabilized with 0.5% Triton X-100 (in 1 × PBS) for 20 min at room temperature. Primary antibodies for staining CTPS2 (ab32087, Abcam), BRCA1 (Santa Cruz, TX, USA) and p-H2AX (ab81299, Abcam) were used at 1:200 for 1 h at room temperature. After three washes with PBS, the sections were incubated with the secondary antibody in PBS with 2% normal serum for 1 h at room temperature, followed by three washes in PBST. Subsequently, cells were stained with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI; a DNA-specific fluorescent dye) for 5–10 min. Stained cells were covered with coverslips and visualized using confocal microscope.

Antibodies used for immunoblotting analyses in this study are listed below: rabbit anti-MCL-1 (1:500, ab32087), rabbit anti-BAX (1:1000, ab32503) were obtained from Abcam; mouse anti-BCL-2 (1:1000, #15071), rabbit anti-BIM (1:1000, #2933), rabbit anti-BCL-XL (1:1000, #2762), rabbit anti-BAK (1:1000, #12105), rabbit anti-AKT (1:1000, #4691), rabbit anti-p-AKT (1:1000, #4060), rabbit anti-mTOR (1:1000, #2983), rabbit anti-p-mTOR (1:1000, #5536), rabbit anti-p-4EBP1 (1:1000, #2855), rabbit anti-4EBP1 (1:1500, #9644), rabbit anti-FOXO3a (1:1000, #12829), rabbit anti-p-FOXO3a (1:1000, #9466) rabbit anti-cleaved PARP (1:1000, #5625) and rabbit anti-cleaved Caspase-3 (1:1000, #9664) were purchased from Cell Signaling Technology; mouse anti-β-actin (1:1000, sc-47778), mouse anti-GAPDH (1:1000, sc-32233) and rabbit anti-PUMA (1:1000, sc-28226) were from Santa Cruz Biotechnology; rabbit anti-p-4EBP1 (1:500, NB100-81769, used in Fig. 1b) and mouse anti-4EBP1 (1:1000, NBP1-47366, used in Fig. 1b) were obtained from Novus Biologicals. The compounds of BH3 mimetics (ABT263 and ABT199) and mTOR inhibitors (BEZ235, AZD8055, and Temsirolimus) were from AbMole Bioscience.

HK-2 cells were thoroughly lysed using RIPA lysis buffer. Total protein in the lysate was assessed using Bradford assay. Equal amounts of proteins were loaded onto sodium dodecyl sulphate polyacrylamide gel. The separated samples were then transferred onto a PVDF membrane. The membrane was further blocked using 5% skim milk and incubated with primary antibodies (MCL-1, Abcam, ab32087, 1:1000), PCNA (Abcam, ab29, 1:1500), Cyclin D1 (Abcam, ab16663, 1:1000), BAX (Abcam, ab32503, 1:1000), Bcl-2 (Abcam, ab182858, 1:1000), Cleaved-caspase-3 (Cell signaling, #9664, 1:1500), NOX1 (Abcam, ab121009, 1:1000), NOX2 (Abcam, ab310337, 1:1500), SOD1 (Abcam, ab51254, 1:1000), SOD2 (Abcam, ab68155, 1:1000), Caspase-3 (Abcam, ab184787, 1:1000), β-Actin (Abcam, ab8226, 1:3000), and GAPDH (Abcam, ab8245, 1:2000), overnight at 4 °C. Further, the blots were washed and incubated with secondary antibody for 1 h at RT. Finally, the bands were visualized using ECL western blotting substrate (Invitrogen, 32109, USA). Western blot images were quantified by using ImageJ (V1.8.0.112, National Institutes of Health, Bethesda, MD), with the density of each band normalized to that of β-Actin or GAPDH (Additional file 2).

BCL1 cells, grown in culture plates, were treated with or without Pt(S-pr-thiosal)2 (0.05 mg/mL) for 12 h. The treated cells were fixed and permeabilized with permeabilization buffer (BD Bioscience) and incubated with antibodies specific for Bcl-2 (11-6992-42, Thermo Fisher Scientific, Cambridge, UK), Noxa (ab13654, Abcam, Cambridge, UK), STAT3 (IC1799G, Novus Biologicals, San Diego, CA, USA), p16 (ab211542, Abcam, Cambridge, UK), p21 (ab188224, Abcam) and p27 (ab215434, Abcam), cyclin D3 (ab28283, Abcam Cambridge, UK), cyclin E (MA5-14336, Thermo fisher scientifics, Waltham, MA, USA), Ki-67 (151212, BioLegend, San Diego, CA, USA), Noxa (ab13654, Abcam) and Mcl-1 (ab32087, Abcam). For staining p16, p21, and p27 cells were additionally incubated with secondary goat anti-mouse IgG FITC (ab6785, Abcam) or donkey anti-rabbit IgG (ab150073, Abcam). Flow cytometry was performed on FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo (Tree Star).

The esophagus tissues were fixed in 10% buffered formalin for 48 hours. The paraffin-embedded 5 μm-thick esophagus tissue sections from different groups were processed for H&E staining. For IHC staining, slides were incubated at 55°C for 2 hours and deparaffinized in xylene and rehydrated through graded alcohols (100%, 95%, 70% alcohol; 2 times 3 minutes in each grade). Slides were rinsed in ddH₂O and then blocked with 0.3% H₂O₂ for 30 minutes to block endogenous peroxidase activity. Heat-induced antigen retrieval was performed in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) using a pressure cooker. Sections were rinsed in PBS and blocked in 10% normal donkey serum, 1% bovine serum albumin and 0.1% Triton X-100. Sections were incubated with primary antibody against Keratin 5 (Abcam, ab52635, 1:500), Mcl1 (Abcam, ab32087, 1:200), p-p65 (Abcam, ab86299, 1:100), caspase3 (Cell Signaling, #9663, 1:500) and Ki67 (Thermo Fisher Scientific, RM-9106-S1, 1:200) overnight at 4°C. Section were rinsed in PBS and incubated with biotinylated secondary antibody and streptavidin-biotinylated horseradish peroxidase complex (Zhongshan, Beijing, China) 1 hour at room temperature. Proteins expression was detected by using Diaminiobenzidine (DAB) as substrate.

All the protein was extracted from cultured cells or tissues by RIPA (Beyotime, Shanghai, China) with protease inhibitor cocktail (Roche, Basel, Switzerland). The concentration of protein was measured by BCA Protein Assay Kit (Pierce, Rockford, IL, U.S.A.). Samples were loaded equally in lanes, resolved using SDS/PAGE (Beyotime, Nantong, China), and transferred on to a nitrocellulose membrane. The membranes were blocked at room temperature with 5% BSA for 1 h, and incubated overnight at 4°C with primary antibodies (anti-MCL1, #ab32087, 1:1000; anti-GAPDH, #ab181603, 1:10000, Abcam, Cambridge, MA, U.S.A.). Consecutively, the secondary antibodies (IgG-HRP, #ab7090, 1:2000, Abcam, Cambridge, MA, U.S.A.) were added and the protein was incubated for another 1.5 h at low temperature shaker. ECL Plus (Life Technology, Shanghai China) was used for protein visualization, and GAPDH was used for quantity normalization of the protein.