Fig. 8. Manipulation of SRPK2 and FGF21 modulates dysregulation of SRSF10-mediated lipogenic pre-mRNA splicing associated with ALD.

A-B. Real-time qRT-PCR analysis of mRNA levels of total lipin 1 and different lipin 1 splicing isoforms in pair-fed and alcohol-fed mice. Lipin 1β/α ratio was calculated using their relative mRNA levels.

C. Positive correlation between hepatic SRPK2 and lipin 1β levels in pair-fed and alcohol-fed mice.

D. Immunoblots for SRSF10 in pair-fed and alcohol-fed mice.

E-F. Immunoblots (E) and densitometric quantification (F) for SRSF10 in alcohol-fed mice that were injected with either AdshRNA control or AdshSRPK2.

G. Immunohistochemistry for SRSF10. Notably, upon chronic-binge alcohol feeding, positive staining for SRSF10 was visualized mainly in the nucleus of hepatocytes in Ad-shRNA control-injected control mice, but the intensity and distribution of SRSF10⁺ hepatocytes were increased in SRPK2-knockdown mice.

H-I. Real-time qRT-PCR analysis of lipin 1, lipin 1β, lipin 1α, as well as lipin 1 β/α ratio in control and SRPK2-knockdown mice.

J. Ethanol exposure enhanced the interaction between SRPK2 and SRSF10 in AML-12 hepatocytes. AML12 cells were transduced with Ad-SRPK2 at an MOI of 5 and subsequently incubated for 24 h with or without ethanol (100 mM) in medium containing 2% FBS. Cell lysates were subjected to immunoprecipitation with an anti-SRPK2 antibody; immunoprecipitated proteins and cell lysates were immunoblotted with indicated antibodies.

K-L. Immunoblots (K) and densitometric quantification (L) for SRSF10 in WT and FGF21-Tg mice.

M-N. Real-time qRT-PCR analysis showed that mRNA levels of lipin 1 and lipin 1β, as well as the lipin 1β/α ratio, were increased by alcohol administration in WT mice, and this induction was attenuated in FGF21-Tg mice.

O-P. Real-time qRT-PCR analysis for skipping of exon 7 (lipin 1α) and inclusion of exon 7 (lipin 1β) revealed that rapamycin treatment decreased lipin 1β induction caused by chronic binge alcohol feeding but did not affect mRNA expression levels of lipin 1α.

Q-R. Real-time qRT-PCR analysis of mRNA levels of lipin 1 and lipin 1 spliced isoforms as well as the lipin 1β/α ratio in WT and FGF21 KO mice.

S. Immunoblots for SRSF10 showed that upon alcohol administration, hepatic SRSF10 was downregulated in AdshRNA-injected FGF21 KO mice and this impairment was rescued by silencing SRPK2.

T-U. Real-time qRT-PCR analysis of mRNA levels of lipin 1, lipin 1β, and lipin 1α, as well as the ratio of lipin 1β/lipin 1α.

The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups.

Images were acquired using 20X and 40X objectives.