Extended Data Fig. 8: IAP is a key downstream carRNA target to rescue aberrant HSPC proliferation and differentiation caused by Tet2 deficiency.
From: RNA m5C oxidation by TET2 regulates chromatin state and leukaemogenesis
a, RT-qPCR quantification for IAP levels in caRNAs isolated from WT or Tet2-KO (Tet2−/−) HSPCs. b, Representative flow cytometry analyses of dCas13d-TET2CD fusion protein and guide RNA transfection efficiency in HSPCs. c, Replating assay of HSPCs transfected with guide RNA targeting the IAP RNA using either the RfxdCas13d fusion protein with TET2 catalytic ___domain (TET2CD) or the inactivated mutant (TET2HxDCD). d, Flow cytometry analyses of HSPC frequency in suspension cultures (day 7) of WT and Tet2−/− HSPCs transfected with guide RNA targeting the IAP RNA using the RfxdCas13d fusion protein with TET2CD or TET2HxDCD. e, Flow cytometry analyses of differentiation (CD11b, day 14) of WT and Tet2−/− HSPCs transfected with guide RNA targeting the IAP RNA using the RfxdCas13d fusion protein with TET2CD or TET2HxDCD. f, Representative flow cytometry analyses of ASO transfection efficiency in HSPCs. g, Replating assay of WT and Tet2−/− HSPCs transfected with control (NC ASO), MERVL-targeting (MERVL ASO) or IAP-targeting (IAP ASO) steric antisense oligos. Comparisons against “NC ASO” group. h, Flow cytometry analyses in suspension cultures (day 7) of the HSPCs frequency of WT and Tet2−/− HSPCs transfected with NC ASO, MERVL ASO or IAP ASO. i, Flow cytometry analyses of differentiation (CD11b, day 14) of WT and Tet2−/− HSPCs transfected with NC ASO, MERVL ASO or IAP ASO. j, Overlaps among differentially expressed genes upon Tet2 KO or the treatment with IAP ASO in HSPCs (total RNA-seq). Barplot depicting the odds ratio of upDEGs following Tet2 KO, in comparison to both upDEGs and downDEGs after treatment with IAP ASO in HSPCs. k, GSEA enrichment analysis conducted using genes downregulated (downDEGs) upon the treatment of ASO in HSPCs as the gene list and expression log2FC upon Tet2 KO in HSPC cells as the rank list. l, Functional enrichment analysis on genes that are upregulated following Tet2 KO and downregulated upon treatment with an ASO targeting IAP. These genes were located near m5C peaks. Bar (a,c,g) or line centre (d,e,h,i) represents mean and whiskers s.d. P values, two-tailed Fisher’s Exact Test (j), two-tailed unpaired t test with Welch’s correction (a,c-e,g-i), two-tailed permutation (k), one-tailed Fisher’s Exact Test (l). NS, P > 0.05. n = 3 biological replicates (a,c-e,g-i). The depicted genome-wide data represent an integration of three biological replicates.
