Fig. 3: IGF2BP3 directly regulates expression of ATF4, PHGDH, and PSAT1 via m6A recognition.
From: m6A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability
A Western blot after IGF2BP3 KD in various AML cell lines. GAPDH was used as a loading control. B Western blot of U937 BP3-mAID cells after treatment with 5’Ph-IAA for the indicated time periods. ACTB was used as a loading control. C Western blot of Molm13 cells after overexpression of wild-type (BP3-WT) or KH3-4 mutated (BP3-KH34) IGF2BP3. D Western blot of Molm13 cells after METTL14 KD. E IGV tracks showing the distribution of m6A (GEO: GSE97408) or IGF2BP3 binding sites (GEO: GSE90639) in target mRNAs. Gray shadow depicts high-confidence m6A regions for qPCR validation in (G) and (H). F RIP assays using an IGF2BP3 antibody were performed in Molm13 cells, followed by qPCR to detect direct binding of IGF2BP3 to target mRNAs at regions indicated in (E). G MeRIP-qPCR was performed in control and METTL14 KD Molm13 cells to detect the change of m6A abundance at indicated regions of ATF4, PHGDH, and PSAT1 transcripts. H Bst DNA polymerase-mediated cDNA extension and qPCR assays to evaluate relative m6A abundance changes at specific sites within ATF4, PHGDH, and PSAT1 transcripts in control and METTL14 KD Molm13 cells. I Control and IGF2BP3 KD Molm13 cells were treated with actinomycin D for indicated time periods and the RNA level of target genes was examined by qPCR. 18S rRNA was used as a loading control. The mRNA half-life (t1/2) was calculated and shown. (J, K) Live cell counting (J) and cytometric analysis of Annexin V+ apoptotic cells (K) in Molm13 cells upon IGF2BP3 KD and ATF4, PHGDH, or PSAT1 overexpression. EV, empty vector. (L, M) Molm13 cells were co-transduced with control or IGF2BP3 shRNAs and ATF4/PHGDH/PSAT1 overexpression vectors and subjected to colony-forming assays (L) and serine level detection (M). Representative results from one of the two independent experiments were shown (A–D). Mean ± SD values are shown. n = 3 biologically independent repeats in (F, G, H, I, K, and M), while n = 2 biologically independent repeats in (J) and (L). Two-tailed student’s t-test (F–H), two-way ANOVA (K, M). Source data are provided as a Source Data file.
