Fig 1: S1P overexpression promotes RCC cell proliferation and migration.Primary RCC cells (“RCC1/RCC2/RCC3”) or A498 cells, bearing the lentiviral S1P expression construct (“OE-S1P”) or empty vector (“Vec”), were established, expression of listed genes in these cells and parental control cells (“Pare”) was shown (A–C and G). Cells were further cultured for applied time periods, CCK-8 OD (H), proliferation (by recording nuclear EdU ratio, D and I), migration, and invasion (“Transwell” assay, E, F, and J) were tested. RCC1 cells bearing SREBP1 shRNA (“sh-SREBP1”) or SREBP1-expressing construct (OE-SREBP1) were established, control cells were transduced with scrambled control shRNA plus empty vector (“Vec+shC”), expression of SREBP1 mRNA and protein was shown (K). Cells were further cultured for applied time periods, viable cell number (L), proliferation (M), and migration (N) were tested similarly. For each assay, n = 5. Data were expressed as the mean ± standard deviation (SD). *P < 0.05 vs. “Vec”/“Vec+shC” group. In this figure, experiments were repeated three times, and similar results were obtained each time.
Fig 2: SREBP1-S1P upregulation in human RCC.Eight (n = 8) pairs of human RCC tumor tissues (“T”) and surrounding normal renal tissues (“N”) were obtained, expression of listed genes was tested by RT-qPCR (A-C) and Western blotting (D, E) analyses, results were normalized and quantified. The TCGA cohort shows relative SREBP1 transcripts in 533 cases of ccRCC tissues (“Primary Tumor”) and 72 cases of normal renal tissues (“Normal”) (F). Kaplan–Meier Survival analyses of SREBP1-low (n = 398, in blue) and SREBP1-high (n = 133, in red) ccRCC patients were shown (G). Data were expressed as the mean ± standard deviation (SD). *P < 0.05 vs. “N” tissues.
Fig 3: S1P silencing or knockout inhibits RCC cell growth.Stable RCC1 cells expressing S1P lentiviral shRNA (“sh-S1P” cells) or the CRSPR/Cas9-S1P-KO-GFP construct (“ko-S1P” cells) were established, control cells were transduced with scrambled control shRNA plus CRSPR/Cas9 empty vector (“sh-C + Cas9-C”); Expression of listed genes was shown (A and B); Cells were then treated with or without PF-429242 (10 μM), and cultured for applied time periods, total cholesterol levels were tested (C); Viable cell number, cell death, proliferation, and migration were tested by CCK-8 (D), LDH release (D), nuclear EdU staining (E) and “Transwell” (F) assays, respectively, and results were quantified.RCC2, RCC3, and A498 cells, expressing S1P lentiviral shRNA (“sh-S1P” cells) or scramble control shRNA (“sh-C”), were established and S1P mRNA expression was shown (G); Cells were further cultured for applied time periods, total cholesterol levels were tested (H); Cell proliferation, migration, and cell death were tested by nuclear EdU staining (I), “Transwell” (J) and medium LDH release (K) assays, respectively. For each assay, n = 5. Data were expressed as the mean ± standard deviation (SD).* P < 0.05 vs. “sh-C + Cas9-C”/“sh-C” group. “n.s.” stands for no statistical difference (C–F). In this figure, experiments were repeated three times, and similar results were obtained each time. Scale bar = 100 μm (E and F).
Fig 4: PF-429242 inhibits subcutaneous RCC xenograft growth in mice.RCC1 xenograft-bearing SCID mice were subjected to i.v. injection of PF-429242 (“PF”, at 10 mg/kg body weight, daily, for 21days) or vehicle control (“Veh”), tumor volumes (A) and mice body weights (D) were recorded every seven days; The estimated daily tumor growth, in mm3 per day, was calculated using the described formula (B). On Day-35 tumors of the two groups were separated through surgery and individually weighted (C). On Day-7 and Day-14, one tumor of each group was isolated and homogenized in tissue lysis buffer, expression of listed genes was shown (E and F). RCC1 xenograft-bearing SCID mice were subjected to intratumoral injection of SREBP1 shRNA lentivirus (“shSREBP1”), S1P shRNA lentivirus (“shS1P”) or scramble control shRNA lentivirus (“shC”), daily for five days, tumor volumes (G) and mice body weights (H) were recorded every seven days; At Day-14, one tumor of each group was isolated and homogenized in tissue lysis buffer, expression of listed proteins was shown (I). Data were expressed as the mean ± standard deviation (SD).*P < 0.05 vs. “Veh”/ “shC” group.
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