Extended Data Fig. 7: The DAMER–ALKBH5-ATF4 axis is a linchpin for metabolic adaptation.
a, MTT cell growth assays of ALKBH5-silenced cells transfected with ATF4 or DAMER under nutrient deficiency. b, Asn concentrations in ALKBH5-silenced cells transfected with ATF4 or DAMER under nutrient deficiency. c, WB analysis of ALKBH5, ATF4, and ASNS levels in the indicated cells. d, MTT cell growth assays of 95D sg-ctrl and DAMER-KO1 cells overexpressing ALKBH5-WT, ALKBH5-H204A, or ALKBH5-Y141A under nutrient deficiency. e, Schematic of N-terminally Flag-tagged full-length ALKBH5 (FL) and its deletion mutants. f, Exogenous RNA pull-down assays validating the interaction between ALKBH5 deletion mutants and DAMER or ATF4 mRNA. g, SELECT assay showing m6A levels at sites 755 and 774 of DAMER in the indicated cells under nutrient deficiency. h, WB analysis of ATF4 levels in 95D sg-ctrl and DAMER-KO1 cells transfected with ATF4-WT or ATF4-A1599G under nutrient deficiency. i, m6A-RIP assay of ATF4 mRNA m6A levels in 95D sg-ctrl cells using the dCasRx-METTL3 system under nutrient deficiency. j, Asn concentrations in 95D sg-ctrl cells using the dCasRx-METTL3 system under nutrient deficiency. k–n, Indicated cells were subcutaneously inoculated in nude mice. k,l, Representative images of xenograft tumours at the end point. Scale bar, 1 cm. m,n, Glc, Gln, and Asn concentrations in the periphery and core tumour tissues. Representative images or tables are shown for Western blots (c,h), RNA pull-down assays (f), and xenograft tumours (k,l). All other experiments were repeated independently three times with similar results. Data in quantitative graphs are presented as mean ± s.d. from n = 3 independent biological replicates (a,b,d,g,i,j,m,n). P values are calculated by two-way ANOVA followed by Tukey’s test (a,b,d,g,j,m,n) or two-tailed unpaired Student’s t-test (i). Source numerical data, precise P values and unprocessed blots are provided.
