Fig 1: SF3B2 is de-fatty acylated by HDAC11.(A) SF3B2 was chosen as a potential substrate after cross-referencing three mass spectrometry datasets. (B) Co-IP of SF3B2 and HDAC11. (C) SF3B2 lysine fatty acylation by Alk14. (D) Quantification of C. (E) SF3B2 K fatty acylation with WT or catalytically dead Y304H HDAC11. (F) Quantification of E. (G) Relative quantification of E. Each condition is normalized by dividing by the +Alk14 signal from its paired control.Each shape in the dot plots represents an independent experiment. Statistics are by paired T-test. *p<0.05, **p<0.01, ns: not significant (p>0.05).
Fig 2: SF3B2 is myristoylated at Lysine 10.(A) Mass spectra indicating K-myr at SF3B2 K10. (B) Western blot assaying K-myr between SF3B2 and K10R in SF3B2 HEK cells. (C) Quantification of B. (D) Relative quantification of B. Each condition is normalized by dividing the K-myr signal from its paired control.Each shape in the dot plots represents an independent experiment. Statistics are by paired T-test. **p<0.01, ns: not significant (p>0.05).
Fig 3: HDAC11 regulates AR-v7 alternative splicing in Hep-G2 cells via SF3B2 de-myristoylation.(A) Western blot indicating HDAC11 overexpression in Hep-G2 cells. (B) qPCR analysis of AR exon junctions in Hep-G2 cells with overexpressed HDAC11. (C) Analysis of AR-v7/AR-FL splicing ratio in Hep-G2 cells with overexpressed HDAC11. (D) AR-v7/FL splicing ratio using minigene in Hep-G2 cells with SF3B2 WT versus K10R. (E) Western blot indicating HDAC11 KD in Hep-G2 cells. (F) qPCR analysis of AR exon junctions in Hep-G2 HDAC11 KD cells. (G) Analysis of AR-v7/AR-FL splicing ratio in Hep-G2 HDAC11 KD cells. (H) AR-v7/FL splicing ratio in Hep-G2 HDAC11 KD cells with overexpressed WT or mutant HDAC11. (I) AR-v7/FL splicing ration in Hep-G2 HDAC11 KD cells with overexpressed WT or K10R SF3B2. Each shape in the dot plots represents an independent experiment. Statistics are by paired T-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant (p>0.05).
Fig 4: In PCa cells, AR splicing is unaffected by HDAC11 overexpression, HDAC11 KD, or SF3B2 de-myristoylation mimetic.(A) Western blot of AR-FL and AR-v7 with HDAC11 overexpression in 22Rv1 prostate cancer cells. (B) qPCR analysis of AR exon junctions with overexpressed HDAC11. (C) Analysis of AR-v7/AR-FL splicing ratio with overexpressed HDAC11. (D) Western blot of AR-FL and AR-v7 with HDAC11 KD in 22Rv1 prostate cancer cells. (E) qPCR analysis of AR exon junctions with HDAC11 KD. (F) Analysis of AR-v7/AR-FL splicing ratio with HDAC11 KD. (G) Diagram of AR-v7 minigene assay. (H) AR-v7/FL splicing ratio using minigene in 22Rv1 cells with SF3B2 WT versus K10R de-myr mimetic. (I) AR-v7/FL splicing ratio using minigene in PC3 cells with SF3B2 WT versus K10R.Each shape in the dot plots represents an independent experiment. Statistics are by paired T-test. *p<0.05, **p<0.01. ns: not significant.
Fig 5: K-myr loss causes cell type specific changes to SF3B2 pre-mRNA binding.(A) Schematic of constitutive AR exons, including cryptic exon 3. Exon 1 (blue) encodes the N-terminal domain, exons 2–3 (pink) encodes the DNA binding domain, exon 4 (green) encodes the hinge domain, and exons 5–8 (yellow) encode the ligand binding domain. (B) RNA-IP of SF3B2-Flag in 22Rv1 prostate cancer cells. (C) RNA-IP of SF3B2-Flag in Hep-G2 hepatocellular carcinoma cells. (D-F) SF3B2 WT or K10R binding in 22Rv1 cells at exon 1 (D), intron 3-exon 3 (E), or intron 7-exon 8 (F). (G-I) SF3B2 WT or K10R binding in Hep-G2 cells at exon 1 (G), intron 3-exon 3 (H), or intron 7-exon 8 (I).Each shape in the dot plots represents an independent experiment. Statistics are by paired T-test. **p<0.01. ****p<0.0001, ns: not significant (p>0.05).
Supplier Page from Sino Biological, Inc. for Human SF3B2 Gene ORF cDNA clone in cloning vector