Ab76424

Whole‐cell extracts were prepared in radioimmune precipitation assay (RIPA) buffer (50 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1% NP‐40, 0.1% SDS, 0.5% sodium deoxycholate, 0.5 mmol/L DTT, 0.5 mmol/L PMSF and 2.5 mmol/L Roche protease inhibitor cocktail). The extracts were subjected to Western blot analysis as described previously.¹¹ Briefly, 20 to 40 μg of total protein were separated using 7‐12% SDS‐PAGE and transferred to PVDF membrane. The membrane containing transferred proteins was blocked by incubating with 5% BSA containing 0.05% Tween‐20 and incubated overnight at 4°C with primary antibody to detect CYCLIN D1 (Abcam, ab134175), c‐MYC (Abcam, ab62928), SURVIVIN (Abcam, ab76424), α‐TUBULIN (Abcam, ab4074), DKK1 (Abcam, ab109416), DKK3 (Abcam, ab186409), β‐ACTIN (Cell Signaling Technology, 4970), CYCLIN D3 (Cell Signaling Technology, DCS22) and PRMT5 (Thermo Fisher, MA1‐25470). After incubation with primary antibody, the membrane was treated with HRP‐conjugated goat anti‐mouse (Amersham Biosciences, NA931) or anti‐rabbit (Amersham Biosciences, NA934V) secondary antibody. Next, proteins were visualized using the ECL detection kit (Amersham, RPN2209) in a Western blot imager (Flurochem E system, proteinsimple).

HUVECs were lysed in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China), and protein concentration was measured by BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Next, membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight 4°C with the primary antibodies as follows: cathepsin C (Abcam, ab199109, 1:1000), p-p38 MAPK (Abcam, ab178867, 1:1000), p38 MAPK (Abcam, ab170099, 1:5000), p-NF-κB p65 (Abcam, ab76302, 1:1000), NF-κB p65 (Abcam, ab32536, 1:10,000), p-IκBα (Abcam, ab133462, 1:10,000), IκBα (Abcam, ab32518, 1:10,000), Bcl-2 (Abcam, ab196495, 1:2000), Bax (Abcam, ab53154, 1:1000), Birc5 (Abcam, ab76424, 1:5000), Cleaved PARP (Abcam, ab32064, 1:10,000), PARP (Abcam, ab191217, 1:1000) and GAPDH (Abcam, ab9485, 1:2500). On the second day, the membranes were incubated with the corresponding secondary antibody (Abcam, ab205718, 1:50,000) for 1.5 h at room temperature. Protein blots were visualized using electrochemiluminescence (ECL; Beyotime, Shanghai, China) method and analyzed by a Bio-Rad imaging system (Bio-Rad, CA, USA).

To validate the results of scRNA-seq analysis, we selected totally 8 highly expressed genes in CSC cluster (n=4) and Cancer cell clusters (n=4). By immunohistochemistry (IHC), we stained sections of 5-µM thickness from the paraffin blocks of 17 CDRCC patients (Supplementary Table 6). According to the immunohistochemical scores, Kaplan-Meier curve was drawn to present the relationship between the expression level and survival time. Second, to verify the possible therapy drugs to CDRCC, we selected 1 CSC-related gene and 4 targeted therapy genes to carry out double immunofluorescence labeling staining to detect the gene expression level in CSC cluster. The following antibodies were used to represent the expression of the selected genes: anti-PARP1 (rabbit, 1:500, Abcam, ab32138), anti-PIGF (rabbit, 1:300, Proteintech, 10642-1-AP), anti-HDAC2 (rabbit, 1:500, Abcam, 32117), anti-FGFR3 (rabbit, 1:200, Abcam, ab137084), anti-BIRC5 (rabbit, 1:500, Abcam, ab76424), anti-PTTG1 (rabbit, 1:1000, Abcam, ab79546), anti-CENPF (rabbit, 1:500, Abcam, ab223847), anti-CDKN3 (rabbit, 1:500, Abcam, ab206314), anti-ATF3 (rabbit, 1:1000, Novusbio, nbp1-85816), anti-PDZK1 (mouse, 1:200, R&Dsystems, af4997), anti-VTN (rabbit, 1:300, Abcam, ab45139), anti-CXCL8 (mouse, 1:500, R&Dsystems, af-208-na)(Figure 4, Supplementary Figure 8).

The tested compounds were provided by Dongdong Sun (Nanjing University of Chinese Medicine). For the treatment, the compounds were dissolved in culture medium and DMSO [with DMSO less than 0.1%(v/v)]. HCT116 colorectal cancer cells were supplied from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 culture medium was provided by Biological Industries (Beit‐Haemek). TUNEL apoptotic commercial kit was produced by Beyotime. Annexin V‐PI kit was purchased from KeyGEN. α‐ketoglutarate as obtained from Jinglai Biotechnology. The commercial kits for ATP, glutamine, glutamate and glutathione were provided by Jiancheng Biotechnology. The ASCT2 inhibitor GPNA was produced by Sigma‐Aldrich. Pifithrin‐α, the selective inhibitor for P53, was obtained from APExBIO. ASCT2 (#ab84903, 1:1000), P53 (#ab26, 1:1000), caspase‐3 (#ab197202, 1:1000), caspase‐7 (#ab69540, 1:1000), cleaved‐caspase‐3 (#ab2302, 1:1000), cleaved‐caspase‐7 (#ab2323, 1:1000), cleaved‐PARP (#ab32064, 1:1000), PARP (ab74290, 1:1000) and survivin (ab76424) were purchased from Abcam.

THP-1 macrophages grown on coverslips were fixed with 4% (w/v) paraformaldehyde, rehydrated with 2% (v/v) fish skin gelatin and permeabilized with 0.2% Triton X-100 prior to incubation with 5% (v/v) goat serum. Subsequently, cells were incubated overnight at 4 °C with an anti-survivin antibody (1:500 dilution; ab76424, Abcam) in PBS containing 1% goat serum. Next, the cells were washed with PBS and incubated for 1 h at room temperature with an Alexa Fluor 488-conjugated secondary antibody (1:100 dilution; Life Technologies, Carlsbad, CA, USA) followed by mounting with ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were acquired on a Leica DM 4000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and captured with a Leica DFC 300 FX camera (Leica Microsystems). Fluorescence intensity was analyzed using ImageJ software (https://imagej.nih.gov/ij/download.html), as described before [41 (link)].

Total protein was isolated and collected from HGSOC cell lines with radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotech, Santa Cruz, CA, USA). The isolated proteins with equal quantity were further separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE; 10% or 12%), followed by transferring onto separate polyvinylidene fluoride membranes (Beijing Solarbio Science& Technology, Beijing, China). The antibodies used for immunoblotting were the following: GAPDH (Cell Signaling Technology, Beverly, MA, USA), SOCS7 (ab224589; Abcam, Cambridge, MA, USA), HuR (ab238528; Abcam), Cyclin D1 (ab16663; Abcam), Survivin (ab76424; Abcam), CDC25B (ab124819; Abcam), and FOXM1 (ab207298; Abcam). After incubating with horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology), the signals were further examined based on an enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA).

Survivin neutralization was performed by adding 20 μg/ml of an anti-survivin antibody (ab76424; Abcam, Milton, Cambridge, UK) for 1 h at room temperature before adding the medium to THP-1 or HepG2 cells. A negative epitope control (Rabbit IgG Isotype Control, Invitrogen) was included in each experiment.