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. 2020 Nov 11;6(46):eabb7426. doi: 10.1126/sciadv.abb7426

Fig. 5. Fly circBoule RNAs regulate protein levels of Hsp60C and Hsc4.

Fig. 5

(A and B) Protein levels of Hsp60C and Hsc4 in testis with (w1118) or without (M-introns KO) circBoule at different time points at 29°C. β-ACTIN was used as endogenous control. (C) Ratio of offspring numbers of w1118, hsp60C-Hsp60C-3xFLAG SCIn (single copy insertion), and hsc4-Hsc4-3xFLAG SCIn males at 29°C. Red (or blue) asterisks and NS indicate statistical significance of difference between w1118 and hsp60C-Hsp60C-3xFLAG SCIn (or hsc4-Hsc4-3xFLAG SCIn) flies. n = males tested. (D and E) Sperm count and sperm with defective nuclear morphology in w1118, hsp60C-Hsp60C-3xFLAG SCIn, and hsc4-Hsc4-3xFLAG SCIn males. Representative images of abnormal sperm nuclear morphology are shown. White triangles indicate mature sperm with abnormally shaped nuclei. n = fly testes analyzed. Scale bars, 20 μm. (F and G) IF of Hsp60C and Hsc4 protein in spermatid bundles using FLAG antibody. circBoule (+), w1118 background; circBoule (−), M-introns KO background. Scale bars, 10 μm. (H) Quantification of spermatid bundles in gDNA-RP, M-introns KO, and Intron3 KO fly testes. Males reared at 29°C were mated with females for 10 days. (I and J) circBoule RNAs promoted ubiquitination of Hsp60C and Hsc4 proteins in fly S2 cells and fly testes, respectively. For S2 cells, circBoule RNAs, HSPs, and ubiquitin were expressed with plasmids. For testes, HSPs were first immunoprecipitated and then analyzed for ubiquitination. Ub, ubiquitin. Flies were 4 days old at 29°C. (K) Ratio of offspring numbers of w1118, Prosα6T+/−, and Rpn11+/− males at 29°C. Red (or purple) asterisks and NS indicate statistical significance of difference between Prosα6T+/− (or Rpn11+/−) and w1118 flies. n = males tested. Data are shown as means ± SD. P values from unpaired Student’s t test, **P < 0.01 and ***P < 0.001.