Journal: Nature Communications

Article Title: The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism

doi: 10.1038/s41467-024-45548-w

Figure Lengend Snippet: a – c Parental and ChREBP overexpressing SNU449 cells were treated with 6-AN (6-aminonicotinamide, 40 μM) for 24 h. Cells were then incubated for 30 min with 11 mM of 13 C 6 -glucose. a Enrichment in (m+6) 6-phosphogluconate from 13 C 6 -glucose ( n = 4 independent experiments). b Enrichment in (m+5) ribose 5-phopshate from 13 C 6 -glucose in response to ChREBP overexpression ( n = 4 independent experiments). c Enrichment in (m+3) ribose 5-phopshate from 13 C 6 -glucose in response to ChREBP overexpression ( n = 4 independent experiments). d De novo nucleotide synthesis from parental and ChREBP overexpressing SNU449 cells incubated 6 h with 11 mM of 14 C 6 -labeled glucose ( n = 4 independent experiments). e Effect of 6-AN treatment (40 μM, 24 h) on ChREBP-mediated increase in hepatocyte proliferation was studied in SNU449 cells. Representative clonogenic assays shown ( n = 7 independent experiments). f Effect of 6-AN treatment (40 μM, 24 h) and dNTPs rescue (100 μM each) on ChREBP-mediated increase in cell proliferation. Cell proliferation index was determined by measuring the % of BrdU positive cells ( n = 4 independent experiments). g Representative Western blot analysis of proteins involved in PPP pathway ( n = 10 biologically independent mice per group). h De novo nucleotide synthesis from 14 C 6 -labeled glucose in ChREBP tumors ( n = 3 biologically independent mice per group). i ChIP experiments measuring ChREBP occupancy at the G6PDH, PGD, TKT and RPIA promoters in ChREBP tumors relative to IgG controls ( n = 4 biologically independent mice per group). j Relative NADPH/NADP ratio in ChREBP overexpressing tumors ( n = 6 biologically independent mice per group). k De novo lipid synthesis from 14 C 6 -labeled glucose in ChREBP tumors ( n = 3 biologically independent mice per group). l Representative Western blot analysis of proteins involved in de novo lipogenic pathways ( n = 10 biologically independent mice per group). m ChIP experiments measuring ChREBP occupancy at the ACC, FAS and SCD1 promoters in ChREBP tumors relative to IgG controls ( n = 4 biologically independent mice per group). n Measurement of SCD1 activity in ChREBP tumors ( n = 3 biologically independent mice per group). o – r FAS expression was knockdown by Crispr/Cas9 in ChREBP overexpressing SNU449 cells (FASi). o Representative Western blot showing FAS deletion in ChREBP overexpressing SNU449 ( n = 3 independent experiments). p De novo lipid synthesis from 14 C 6 -labeled glucose. SNU449 cells were incubated 6 h with 11 mM of 14 C 6 -labeled glucose ( n = 3 independent experiments). q Representative clonogenic assays shown after FAS silencing ( n = 3 independent experiments). r Effect of FAS silencing and oleate supplementation (50 μM) on ChREBP-mediated increase in cell proliferation. Cell proliferation index was determined by measuring the % of BrdU positive cells ( n = 3 independent experiments). All error bars represent mean ± SEM. a – d , f , h , k , n , p , r Statistical analyses were made using two-way ANOVA and Tukey’s multiple-comparisons test. i , j and m Statistical analyses were determined by unpaired two-sided Student’s t test. Source data are provided as a file.

Article Snippet: ChREBP (Novus, NB400-135, 1/1000 e ), SCD1 (Cell Signaling, C12H5, 1/1000 e ), LPK (Abcam, ab6191, 1/1000 e ), ACC (Cell Signaling, 3662, 1/1000 e ), FAS (Cell signaling, C20G5, 1/1000 e ), G6PDH (Cell Signaling, 12263, 1/1000 e ), GAPDH (Santa Cruz Biotechnology, FL-335, 1/1000 e ), PKM2 (Cell Signaling, 4053, 1/1000 e ), GOT1 (Cell Signaling, 344423, 1/1000 e ), G6PDH (Cell Signaling, 8866, 1/1000 e ), GOT2 (Novus Biologicals, NBP1-47469, 1/500 e ), HK2 (Cell Signaling, 2106, 1/1000 e ), PGD (Novus Biologicals, NBP1-31589, 1/1000 e ), RPIA (Novus Biologicals, NBP1-86214, 1/1000 e ), ATP-Citrate Lyase (Cell Signaling, 4332, 1/1000 e ), SCD1 (Cell Signaling, 2794, 1/1000 e ), SLC1A5 (Novus Biologicals, NBP1-89327, 1/1000 e ), Gls1 (Novus Biologicals, NBP1-89766, 1/1000 e ), Gls2 (Novus Biologicals, NBP1-76544, 1/1000 e ), GPT (Novus Biologicals, NBP1-89111, 1/500 e ), CAD (Cell Signaling, 11933, 1/1000 e ), P-CAD Pser1859 (Cell Signaling, 12662, 1/1000 e ), UMPS (Novus Biologicals, NBP1-85896, 1/1000 e ), CTPS1 (Novus Biologicals, NBP1-52892, 1/1000 e ), PRPS2 (Novus Biologicals, NBP1-31435, 1/1000 e ), p(S473)-AKT (Cell signaling, D9E, 1/1000 e ), Akt (Cell Signaling, 9272, 1/1000 e ), P-p70S6K (Cell Signaling, 108D2, 1/1000 e ), p70S6K (Cell Signaling, 49D7, 1/1000 e ), p-GSK3β (Cell Signaling, D17D2, 1/1000 e ), GSK3β (Cell Signaling, 27C10, 1/1000 e ), PTEN (Cell Signaling, 9552, 1/1000 e ), PI3 Kinase p85α (Cell Signaling, 13666, 1/1000 e ), Afp (Cell Signaling, 4448, 1/1000 e ), KI67 (Cell Signaling, 9449, 1/1000 e ), Cyclin D1 (Cell Signaling, 2922, 1/1000 e ).

Techniques: Incubation, Over Expression, Labeling, Western Blot, Activity Assay, Expressing, Knockdown, CRISPR