Ab79546

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 cell lysate was created by treating the cells with RIPA buffer from Epizyme in Shanghai, China, and the amount of protein was measured using the BCA Protein Quantification Kit. A 30 μg protein sample was subjected to electrophoretic separation on a 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were moved to PVDF membranes. Following incubation with a 5% skim milk solution in TBS-T for four hours at room temperature, primary antibodies against TEAD1 (diluted 1:2000, purchased from Abcam, catalog number ab133533, UK) and HIF-1α (diluted 1:2000, acquired from Abcam, catalog number ab79546, UK) were left overnight at 4°C. To ensure equal loading of protein samples, an anti-β-actin antibody (diluted at 1:5000, sourced from Abcam, catalog number ab6275, United Kingdom) was employed. After washing the membranes three times with TBS-T for 15 minutes each, they were then incubated with secondary antibodies (diluted at 1:10,000; Westang) conjugated to horseradish peroxidase for 45 minutes at room temperature. Protein bands were visualized using a developing solution (Vazyme, Nanjing, China) and exposed to X-ray film.

The A549 cells were seeded in six-well plate (5 × 10⁵ per well). After 24 h, either miCtrl or miR (final concentration at 50 nM) were transfected with lipofectamine 2000, respectively. Two days later, the cell lysates were harvested and the total protein was extracted with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of total proteins was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (30 µg total proteins), either from miCtrl or miR treated cells, were loaded and separated on SDS-PAGE gel electrophoresis. The proteins were transferred to membrane and incubated with antibodies (Anti-ILF2 antibody, 1:3000, ab154791; anti-Securin antibody, 1:5000, ab79546; anti-PTGR1 antibody, 1:2000, ab181131; anti-AKR1C1/AKR1C2 antibody, 1:3000, ab179448; anti-PCNA antibody, 1:3000, ab92552; anti-Peroxiredoxin 3 antibody, 1:10000, ab128953; anti-GAPDH antibody, 1:10000, ab128915; Abcam, Cambridge, MA, USA). After incubation with secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), 1:4000, ab97051, Abcam), the images were acquired by using chemiluminesence detection.