Fig 1: ZBTB18 SF-Nte controls lipid uptake via hypoxia pathway and affects cellular metabolism(A) Real-time qPCR of FABP7, an HIF1A target involved in lipid metabolism and trafficking, in GBM-derived BTSC233 cells on ectopic expression of ZBTB18 SF-Nte or ZBTB18 SF-mut; 18S was used as a housekeeping gene. n = 3; *p < 0.05.(B and C) Microphotographs (B) and relative quantification (C) showing the uptake of fluorescent lauric acid compound (Bodipy-C12) by GBM-derived BTSC475 cells expressing ZBTB18 SF-Nte or ZBTB18 SF-mut. Values represent mean ± SD, n = 4; ***p < 0.001, n.s. not significant, according to unpaired t-test (scale bar = 100 µm).(D and E) Microphotographs (D) and relative quantification (E) of LD labeled with Bodipy TMR-X in GBM-derived BTSC475 cells expressing ZBTB18 SF-Nte or ZBTB18 SF-mut. Values represent mean ± SD, n = 4; ***p < 0.001, according to unpaired t-test (scale bar = 100 µm).(F)Real-time qPCR of FABP7 and EGLN3 in GBM-derived BTSC475 cells on ectopic expression of ZBTB18 SF-Nte or ZBTB18 SF-mut, with or without treatment with hypoxia pathway inhibitor, LW6. 72 h on transduction cells were treated with LW6 for an additional 16 h before harvesting. TBP was used as a housekeeping gene. Representative experiment, number of repetitions: 3.(G and H) Microphotographs (G) and relative quantification (H) showing the uptake of fluorescent lauric acid compound (Bodipy-C12) by GBM-derived BTSC475 cells expressing ZBTB18 SF-Nte or ZBTB18 SF-mut, with or without treatment with hypoxia pathway inhibitor, LW6 (scale bar = 100 µm). Cells were treated as in (F). Values represent mean ± SD, n = 4; ***p < 0.001, n.s. not significant, according to unpaired t-test.(I) ATP production rate in GBM-derived BTSC475 cells expressing ZBTB18 SF-Nte or ZBTB18 SF-mut, with or without treatment with hypoxia pathway inhibitor, LW6. Cells were treated as in (F). Values represent mean ± SD, n = 5.(J) Energetic map based on the ATP production basal rates of GBM-derived BTSC475 cells expressing ZBTB18 SF-Nte or ZBTB18 SF-mut, with or without treatment with hypoxia pathway inhibitor, LW6. Cells were treated as in (F). Values represent mean ± SD, n = 5. See also Figure S7.
Fig 2: ZBTB18 cleavage is regulated by a specific amino acidic sequence and requires a PEST sequence(A) Schematic representation of ZBTB18 protein structure in correspondence to the CAPN cleavage site; amino acids in red are those substituted by site-directed mutagenesis.(B and C) Western blot analysis (B) and corresponding quantification (C) of SNB19 cells transduced with empty vector (EV), wild-type ZBTB18 FL (ZBTB18 FL-wt), or ZBTB18 FL with mutated CAPN cleavage site (NLV-mut). ZBTB18 FL (wt or NLV-mut) was detected with an anti-HA antibody. As the HA tag (at the Cte) is not retained in the cleaved form short form, an anti-FLAG antibody (rabbit, Cell Signaling) was used to detect the cleaved fragment.(D and E) Western blot analysis (D) and relative quantification (E) of SNB19 cells on ectopic expression of ZBTB18 FL, ZBTB18 FL without the PEST1 sequence (FL-?PEST1), or ZBTB18 FL with mutated CAPN cleavage site and deleted PEST1 sequence (FL-?PEST1-NLV). Ectopic ZBTB18 variants were detected with an FLAG antibody (mouse, Sigma). The lower molecular weight of FL and SF proteins from FL-?PEST1 and FL-?PEST1-NLV is owing to the absence of the PEST1 region. See also Figure S4 and S5.
Fig 3: CircTP63 promotes HCC progression by regulation of the miR-155-5p/ZBTB18 axis. a, b mRNA and protein expression levels of ZBTB18 in SK-hep1 and SNU-387 cells transfected with si-NC, si-circTP63#1, si-circTP63#1 + miR-155-5p inhibitor or si-circTP63#1 + pcDNA3.1-ZBTB18 was evaluated using qRT-PCR and western blotting assays. c Pearson correlation analysis between circTP63 and ZBTB18 expressions in 90 pairs of HCC tissues. d, e CCK8 and colony formation assays were used to assess the proliferation of SK-hep1 and SNU-387 cells transfected with si-NC, si-circTP63#1, si-circTP63#1 + miR-155-5p inhibitor or si-circTP63#1 + pcDNA3.1-ZBTB18. f, g Transwell assays were used to assess the migration and invasion of SK-hep1 and SNU-387 cells transfected with si-NC, si-circTP63#1, si-circTP63#1 + miR-155-5p inhibitor or si-circTP63#1 + pcDNA3.1-ZBTB18. Data are representative of three independent experiments and shown as mean ± SD., **P < 0.01, compared to the si-NC group; ## P < 0.01, compared to the si-circTP63#1 group
Fig 4: ZBTB18 N-terminal short form localizes in the cytoplasm and no longer functions as a tumor suppressor(A) Western blot analysis of GBM-derived BTSC233 (left) and SNB19 (right) cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. Ectopic proteins were detected with a total ZBTB18 antibody (Sigma).(B and C) Western blot analysis, with anti-FLAG antibody (mouse, Sigma), of nuclear and cytoplasmic fractions of BTSC233 (B) and SNB19 cells (C) on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte.(D) Microphotographs showing SNB19 cells labeled with Flag-ZBTB18 on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte (scale = 100µm).(E) Microphotographs showing the result of a migration assay in SNB19 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte (scale = 100µm).(F) Caspase 3/7 cleavage assay showing apoptotic cells in SNB19 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. Values represent mean ± SD, n = 3; **p < 0.01, n.s. not significant, according to unpaired t-test.(G) Boyden chamber invasion assay showing invading cells in SNB19 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. Values represent mean ± SD, n = 4; **p < 0.01, n.s. not significant, according to unpaired t-test.(H) Proliferation assay (EdU labeling) in SNB19 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. Values represent mean ± SD, n = 4; **p < 0.01, ***p < 0.001, according to unpaired t-test.(I) Real-time qPCR of selected genes in SNB19 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. Cells were harvested 72 h on transduction. 18S was used as a housekeeping gene. Values represent mean ± SD, n = 3; *p < 0.05, **p < 0.01, according to unpaired t-test.
Fig 5: ZBTB18 SF-Nte retains the ability to bind CTBP1/2, preventing them to enter the nucleus(A) Experimental flow chart of the SILAC-based quantitative MS analysis on SNB19 cells labeled using Arg0/Lys0 (low, L), Arg6/Lys4 (medium, M), and Arg10/Lys8 (high, H) and transduced with pCHMWS-EV, pCHMWS-FLAG-ZBTB18 FL, or pCHMWS-FLAG-ZBTB18 SF-Nte, respectively.(B) Summarizing table showing the results of the SILAC MS.(C) Schematic representation of ZBTB18 SF-Nte; in the highlighted CTBP1/2 binding site, amino acids in red are those substituted by site directed mutagenesis.(D) Western blot analysis of CTBP2-ZBTB18 co-immunoprecipitation experiments using a FLAG (rabbit, Cell Signaling) antibody in SNB19 cells expressing ZBTB18 SF-Nte, or ZBTB18 SF-mut. Western blots were performed with mouse anti-CTBP2 or mouse anti-FLAG antibodies (mouse, Sigma).(E) Western blot analysis of nuclear and cytoplasmic fractions of BTSC233 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. ZBTB18 FL and SF were detected with anti-FLAG antibodies (mouse, Sigma).(F) Western blot analysis of nuclear and cytoplasmic fractions of SNB19 cells on ectopic expression of ZBTB18 FL, ZBTB18 SF-Nte, ZBTB18 FL-mut, or ZBTB18 SF-mut. ZBTB18 FL and SF were detected with anti-FLAG antibodies (mouse, Sigma).(G and H) Microphotographs (G) and relative quantification (H) showing CTBP2 immunolocalization in SNB19 cells on ectopic expression of ZBTB18 FL or ZBTB18 SF-Nte. Values represent mean ± SD, n = 5; ***p < 0.001, n.s. not significant, according to unpaired t-test. Scale bar = 100µm.
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