Fig. 4: FAM84A is a downstream target of ALKBH5 in colorectal CSCs.
From: Targeting of the m6A eraser ALKBH5 suppresses stemness and chemoresistance of colorectal cancer
A Dot blot assay of CSC28 with ALKBH5 knockdown and overexpression. B Work-flow for systematic identification of ALKBH5 downstream targets. C Top enriched differential pathways in ALKBH5-knockdown versus control CSC28 cells by RNA-seq. D The top enriched motifs based on m6A peaks in ALKBH5-knockdown CSC28 cells and control cells. P values for m6A motif were derived using the Fisher’s exact test algorithm in HOMER software (upper panel). Differential m6A methylated peaks after ALKBH5 knockdown or overexpression, as determined by MeRIP-seq (lower panel). E Top differential enriched pathways in ALKBH5-knockdown versus control CSC28 cells by Ribo-seq. F Integrated analysis of RNA-seq, MeRIP-seq and Ribo-seq datasets (left panel). The correlation between ALKBH5 and FAM84A mRNA in TCGA (Pearson, two-sided) GEPIA (Pearson, two-sided) datasets and foldchange in RNA-seq (Student’s t-test, two-sided) and Ribo-seq (Student’s t-test, two-sided) (middle panel). The correlation between ALKBH5 and FAM84A mRNA in GSE39582 (Pearson, two-sided) and GSE17536 (Pearson, two-sided) (right panel). G UCSC snapshots of MeRIP-seq reads of FAM84A. Normalized to RNA input. H m6A abundance on FAM84A as revealed by MeRIP-qPCR. Schematic design of MeRIP-qPCR assay (left panel). m6A modification on FAM84A as determined by MeRIP-qPCR (For POP66, n = 3; For CSC28, n = 6, each dot represents an independent sample. One-way ANOVA) (middle panel). m6A modification on FAM84A after overexpression of WT ALKBH5 (A5-OE) or mutant ALKBH5 (A5H204A) in CSC (n = 3, each dot represents an independent sample. One-way ANOVA) (right panel). I Direct Binding between ALKBH5 and FAM84A mRNA, as determined by RIP-qPCR (n = 3, each dot represents an independent sample. Student’s t-test, two-sided). J FAM84A expression as determined by qPCR (n = 3, each dot represents an independent sample. One-way ANOVA). K m6A levels of FAM84A mRNA (left panel), the binding of FAM84A mRNA to ALKBH5 (middle panel) and FAM84A mRNA expression (right panel) in WT and ALKBH5-cKI mice tumor as revealed by MeRIP-qPCR (n = 3, each dot represents an independent mouse. Student’s t-test, two-sided), ALKBH5-RIP-qPCR (Presented data is representative of 3 independent biological replicates. Student’s t-test, two-sided) and qPCR (n = 3, each dot represents an independent mouse. Student’s t-test, two-sided), respectively. The diagram is created in BioRender. Chou, H. (2025) https://BioRender.com/csvnp3n. L Schematic diagram of site-specific RNA targeting using dCas13b-ALKBH5 fusion proteins with gRNA close to the target site. M Establishment of dCas13b-ALKBH5 system in colorectal CSCs as determined by western blot (left panel). FAM84Am6A modification levels (right panel) in colorectal CSCs co-transfected with dCas13b-ALKBH5 and the gRNA (For POP66, n = 4; for CSC28, n = 3, each dot represents an independent sample. Student’s t-test, two-sided). Centers and error bars represent mean and Standard deviation, respectively.
