Fig 2: Measurement of PIP regioisomers in immortalized mouse embryonic fibroblasts (MEFs).a–c MEFs were treated with 1 µM VPS34-IN, 200 nM Apilimod, or vehicle (DMSO) for 90 min, then PIPs were extracted and analyzed using SFC-MS/MS system. a The content of individual PIPs in MEFs (1 × 106 cells) treated with 1 µM VPS34-IN, 200 nM Apilimod or vehicle (DMSO) (n = 3). b, c MRM chromatogram [987.6 ? 605.6 (b) and 1117.6 ? 627.6 (c)] of extracts from MEFs (1 × 106 cells) treated with VPS34-IN, Apilimod or vehicle (DMSO). d–g MEFs were treated with 10 µM PAO, 1 µM A23187, or vehicle (DMSO) for 30 min, and then PIPs were extracted and analyzed using SFC-MS/MS system (see “Methods”). d, e The content of individual PIPs in MEFs (1 × 106 cells) treated with 10 µM PAO, 1 µM A23187 or vehicle (DMSO) (n = 4). f, g Overlay of MRM chromatogram [987.6 ? 605.6 (f) and 1117.6 ? 627.6 (g)] of extracts from MEFs (1 × 106 cells) treated with PAO, A23187, or vehicle (DMSO). h Contents of individual PIPs in MEFs (1 × 106 cells) transfected with the indicated siRNA (n = 3). PIPs were extracted at 48 h after transfection. i, j Intracellular 38:4-PI(3,4,5)P3 content (i) and species (j) in MEFs. MEF cells cultured in complete medium were serum-starved for 12 h (starved) and then treated with 10% FBS for 10 min (stimulated). PIPs were extracted from MEFs at each step and analyzed using SFC-MS/MS system. Data were collected using a QTRAP4500 mass spectrometer. Values are mean ± s.e.m. Data were analyzed by one-way ANOVA with Dunnett’s test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs vehicle (DMSO). Data are from one set of experiments.

Fig 3: 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.

Fig 4: Cezanne promoted autophagy through PIK3C3 and PIK3C2A.A–D IF assay to compare the PI3P production. The fluorescence intensity of GST-2×FYVE labeled PI3P increased in Cezanne OE cells. Quantification of the average intensity of indicated proteins was analyzed by ImageJ. Mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, vs. CT. Scale bars: 20 µm. E, F knockdown of PIK3C3 and PIK3C2A inhibited Cezanne-induced autophagy in cells treated with 5 µM Rapamycin for 4 h.

Fig 5: Cezanne deubiquitinates PIK3C3 at K322.A Immunoblot analysis of lysates from Hek 293T cells transfected with Cezanne, Myc-tagged PIK3C3 (Myc-PIK3C3) and HA-tagged ubiquitin (HA-Ub) or HA- K11/48/63-Ub followed by CO-IP with anti-Myc showed Cezanne would deubiquitinate the K48-linked ubiquitin on PIK3C3. B Immunoblot analysis of lysates from Hek 293T cells transfected with flag-tagged Cezanne(flag-Cezanne) or flag-Cezanne CH and Myc-PIK3C3, HA-Ub followed by CO-IP with anti-Myc showed mutation of Cezanne could not deubiquitinate PIK3C3. C Immunoblot analysis of lysates from Hek 293T cells transfected with Myc-PIK3C3 or Myc-PIK3C3 fragment followed by CO-IP with anti-Cezanne showed the V2 fragment was shown to be the only motif interacting with Cezanne. D Immunoblot analysis of lysates from Hek 293T cells transfected with Cezanne, Myc-PIK3C3 fragment, and HA-K48-Ub followed by CO-IP with anti-Myc indicated that Cezanne would deubiquitinate the K48-linked ubiquitin of V2 fragment of PIK3C3. E Immunoblot analysis of lysates from Hek 293T cells transfected with Cezanne, Myc-PIK3C3, or lysine-mutation PIK3C3 and HA-K48-Ub followed by CO-IP with anti-Myc to analyze the ubiquitination of constructed lysine-mutation PIK3C3.