Figure 3.

Osmotic and oxidative stress impair TDP-43 splice activity. A, HEK293E WT (−)– or shTDP-43–expressing cells were transfected with a CFTR exon 9 mini gene expression vector. After exposure to sorbitol (sorb) or sodium arsenite (ars) for 1 or 3 h or to control medium (−), RNA and RIPA-soluble proteins were extracted. RNA was analyzed with RT-PCR using specific primers for CFTR mini gene, SKAR α and β isoforms, HDAC6, and the housekeeping gene ActB. CFTR exon 9–included (+ exon 9) and excluded (− exon 9) products, as well as SKAR α (exon 3 included) and β (exon 3 excluded) isoforms are labeled. *, nonspecific splice band (top). RIPA lysates were subjected to Western blot analysis with antibodies against TDP-43, SKAR, HDAC6, and phospho-eIF2α as stress induction control and GAPDH as loading control (bottom). B, quantification of CFTR exon 9 exclusion/inclusion ratio (top bar graph), SKAR exon 3 inclusion/exclusion ratio (middle bar graph), and HDAC6 mRNA level normalized to ActB and control conditions (bottom bar graph) from three independent experiments as in A. Data represent the mean ± S.E. (error bars): *, p ≤ 0.05; **, p ≤ 0.005.