FIGURE 3.

Disruption of lin-4 miRNA function contributes to up-regulation of lin-14 mRNA in worms shifted off of food. Wild-type (WT), lin-4(e912) [lin-4(−)], and lin-14(n355) [lin-14(gf)] worm strains were cultured on food for 20 h and then shifted to plates with (24+) or without (24−) food for 4 h (A) RNA was collected at the 24-h time point and used for Agarose Northern blot analyses to detect lin-14, lin-28, lin-41, and actin mRNAs and 18S rRNA and PAGE Northern blot analyses of lin-4 miRNA and control 5.8S rRNA. (B) The mRNA levels of lin-14, lin-28, lin-41, and actin were normalized to 18S rRNA, and the ratio of mRNA levels detected in 24− versus 24+ was calculated for each worm strain. Quantification is the average from three or more independent experiments. Error bars represent SEM and *P < 0.05 is shown for the mutant compared to the WT strains. (C) RNA from WT worms was subjected to PAGE Northern blot experiments to detect lin-4 miRNA and control 5.8S rRNA levels. RNA from four independent experiments was analyzed on the same blot (see Supplemental Fig. 2B). The lin-4 miRNA levels were normalized to 5.8S rRNA and the values from 24+ and 24− were graphed as the average from the four independent experiments and error bars represent SEM. (D) Protein extracts were collected at the 24-h time points and used for Western blot analyses to detect LIN-14, ACTIN, and control TUBULIN proteins. Additionally, 4-h and 20-h time points are shown for WT as well as a 4-h time point from worms null for LIN-14 protein expression [lin-14(−)] [lin-14(n356)(n540)] (Reinhart and Ruvkun 2001). Two different exposures are shown for the LIN-14 Western to demonstrate the absence of a specific LIN-14 band in the lin-14(−) null control worms. (E) The protein levels of LIN-14 and ACTIN were normalized to TUBULIN and the ratio of LIN-14 and ACTIN detected in 24− versus 24+ was calculated for each worm strain. Quantification is the average from three or more independent experiments. Error bars represent SEM.