Fig 2: Expression of PTEN, PIK3C2A, ITPA and BCL3 in ccRCC and their relationship with OS and DFSIHC analysis on tissue microarrays and Kaplan-Meier survival curves were constructed to verify the relationship between PTEN A., PIK3C2A B., ITPA C. and BCL3 D. expression with regard to OS and DFS. (A, B) Negative expression of PTEN and PIK3C2A were correlated with shorter OS and DFS time and worse prognosis. (C, D) Positive expression of ITPA and BCL3 were correlated with shorter OS and DFS time and worse prognosis. Green and blue lines indicated positive and negative expression groups, respectively. P <0.05 was considered to be statistically significant.

Fig 3: PIK3C2A is not a component of the TACC3–chTOG–clathrin–GTSE1 complex. (A,B) Representative confocal micrographs of mitotic and interphase HeLa cells expressing GFP–PIK3C2A and either (A) mCherry–CLTA (mCherry–LCa) or (B) mCherry–TACC3. (C) Representative widefield micrographs of parental HeLa and PIK3C2A-null (PIK3C2A-/-) cells stained for tubulin and either CHC, TACC3, chTOG or GTSE1 (red; protein of interest, POI). (D) Representative widefield micrographs of parental HeLa and PIK3C2A-/- cells treated with GL2 (control) or PIK3C2A siRNA, stained with an anti-PIK3C2A antibody (Proteintech; green) and an anti-tubulin antibody (red). DNA was stained with DAPI (blue). Scale bars: 10 µm.

Fig 4: Survival analysis with SurvExpress (n=468)A, B. Low expression of PTEN and PIK3C2A were correlated with high risk, poor prognosis and shorter OS time. C, D. High expression of ITPA and BCL3 indicated high risk, poor prognosis and shorter OS time. Kaplan-Meier survival curves were also constructed to reveal the relationship between predicted risk of ccRCC patients and the OS time. The results showed that patients with high risk had a significantly shorter OS time than those with low risk (A-D). Green and red lines indicated low- and high-risk groups, respectively. P <0.05 was considered to be statistically significant. Cens: Censored; Event: Death; Prog. Idx.: Prognosis Index.

Fig 5: Cezanne promotes the transcription of PIK3C2A in a POLR2A-dependent way.A The effect of PIK3C3 and PIK3C2A on PI3P production. B PIK3C2A is highly expressed in Cezanne OE PC9 cells. Mean ± SD, n = 3. ****P < 0.0001, vs. CT. C the co-expression analysis of Cezanne and PIK3C2A in the GEPIA database. D MG132 (20 µM) and CQ (40 µM) fail to rescue the decreased expression of PIK3C2A in Cezanne knockdown cells. E, F the mRNA of PIK3C2A elevated in Cezanne OE PC9 and HCC827 cells. G The screen of possible transcription factor of PIK3C2A using hTFtarget database, GEPIA, and LCMS data. H, I co-expression analysis of CTCF (H), POLR2A (I) with PIK3C2A in the GEPIA database. J the LCMS/MS data showed the peptide of POLR2A acquired from immunoprecipitated with Cezanne antibody. K CHIP-seq data from Cistrome Project shown in the UCSC browser, that only POLR2A could bind to the transcription start site of PIK3C2A. L CO-IP assay showed the interaction between Cezanne and POLR2A. M CHX degradation assay showed that Cezanne stabilized POLR2A. N CO-IP assay immunoprecipitated with anti-HA antibody indicated that Cezanne contributed to the deubiquitination of K48-linked ubiquitin. O, P knockdown of POLR2A induced a decreased expression in Cezanne OE PC9 and HCC827 cells.