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. Author manuscript; available in PMC: 2015 Nov 6.
Published in final edited form as: Mol Cell. 2014 Oct 9;56(3):376–388. doi: 10.1016/j.molcel.2014.09.011

Figure 4. miR-14 targets inositol 1,4,5-trisphosphate kinase 2 to regulate autophagy during salivary gland death.

Figure 4

(A and B) Salivary glands dissected from wandering larvae (A) and fat bodies dissected from feeding larvae (B) expressing mCherry-Atg8a in all cells, and ip3k2 knockdown specifically in GFP-marked clone cells (hsflp/w; pmCherry-Atg8a/UAS-ip3k2IR; act<FRT, cd2, FRT>Gal4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta.

(C) Samples from ip3k2 mutant animals (ip3k2 Δ1/Y; +/miR-14Δ1), n = 21 (left), miR-14 null mutant animals (ip3k2 Δ1/w; miR-14Δ1/miR-14Δ1), n = 26 (middle), and ip3k2, miR-14 double mutant animals (ip3k2 Δ1/Y; miR-14Δ1/miR-14Δ1), n = 50 (right), analyzed by histology for the presence of salivary gland material (blue dotted circle) 24 h after puparium formation.

(D) Quantification of data from (C). Data are represented as mean. Statistical significance was determined using a Chi-squared test.

(E) mCherry-Atg8a was expressed in ip3k2 mutant animals (ip3k2 Δ1/Y; miR-14Δ1/pmCherry-Atg8a), miR-14 null mutant animals (ip3k2 Δ1/w; miR-14Δ1/miR-14Δ1, pmCherry-Atg8a), and ip3k2, miR-14 null double mutant animals (ip3k2 Δ1/Y; miR-14Δ1/miR-14Δ1, pmCherry-Atg8a). Salivary glands were dissected 14 h after puparium formation for analyses of mCherry-Atg8a puncta.

(F) Quantification of data from (E). Data are represented as mean +/− SEM; n ≥ 16. Statistical significance was determined using a Student’s t-test. Salivary glands and fat bodies were all stained with Hoechst (blue). Scale bars represent 50 μm.

(G) Protein extracts from salivary glands dissected from wandering larvae of control (w; miR-14Δ1/+; tub-GFP-ip3k2 3′UTR/+) and miR-14 null mutant (w; miR-14Δ1/miR-14Δ1; tub-GFP-ip3k2 3′UTR/+) animals expressing a GFP sensor containing either the wild type ip3k2 3′UTR or a mutated miR-14 binding site in the 3′UTR, and analyzed by immuno-blot with anti-GFP antibody.

(H) Quantification of data from (G). Wild type and mutated sensors are normalized to Beta-tubulin and plotted relative to their respective controls +/− SEM; n’s = 3. Statistical significance was determined using a Student’s t-test.