miR-58 family mutants show defective apoptotic response following irradiation. (A) mir-80; mir-58.1 and mir-80; mir-58.1; mir-81-82 mutants show decreased germline apoptosis following IR, while mir-81-82 double and mir-80; mir-81-82 triple mutant show an increase in IR-induced apoptosis levels. RNAi of T07D1.2, a gene largely deleted in nDf54 deficiency that covers miR-81 and miR-82 locus, induces germline apoptosis following IR. Empty vector was used as control RNAi. Synchronized animals in L4 larval stage were irradiated, and DNA damage–induced germline apoptosis was quantified 24 h after irradiation. Error bars, SEM from three biological replicates (20 worms were scored per experiment); asterisk, P-value for a paired t-test comparing the mutants/T07D1.2 RNAi to the WT/empty vector control (*P < 0.001 IR samples). (B) Apoptotic clearance is not impaired in the mir-80; mir-58.1; mir-81-82 quadruple mutant. Time of corpse persistence was assayed by measuring the time from the corpse's first appearance until it was no longer visible. Average persistence and SD are denoted below the chart. (C) Transcriptional up-regulation of egl-1 following IR is normal in the various miR-58 family mutants. mRNA was extracted 12 h after irradiation of L4 larvae, and egl-1 levels were assayed by quantitative real-time PCR. mRNA levels for each sample were normalized to three housekeeping genes—pgk-1, cdc-42, and Y45F10—and the fold induction was calculated relative to wild-type untreated worms. Error bars, SEM (n = 2). (D) Levels of MPK-1 activation (phosphorylation) differ between various miR-58 family mutants. Protein was extracted from irradiated and nonirradiated young adult worms, and equal amounts were loaded onto SDS-PAGE gels. Total MPK-1 was detected with an anti-ERK-antibody, activated MPK-1 was detected with an anti-P-ERK antibody, and a-tubulin was used as a loading control. The ratio of activated MPK-1 (P) to total MPK-1 (T) normalized against wild-type nonirradiated worms is shown for the MPK-1A and MPK-1B isoforms.
