Given that both organelle clearance and autophagic degradation function were impaired, we wondered whether the failure of organelle clearance in HSF4
del42 lens fibers was due to defective autophagic degradation. To address this issue, autophagy was induced by exogenous stimuli (e.g., rapamycin), and the organelle clearance in HSF4
del42 lenses was detected. The p3 WT and HSF4
del42 lenses were dissected carefully and cultured ex vivo for 24 hours. The lenses in ex vivo models were treated with 10 µM rapamycin for 12 hours to induce autophagy and subjected to immunoblotting. The elevated LC3-II levels and decreased p62 levels demonstrated an obvious induction of autophagy in WT and HSF4
del42 lenses treated with rapamycin (
Fig. 3A). Although rapamycin treatment did not alter the levels of the membrane-bound organelle markers GM130, GRP78, and Tomm20 in WT lenses, it indeed reduced the GM130, GRP78, and Tomm20 levels in HSF4
del42 lenses (
Fig. 3A). Unexpectedly, the nuclear envelope component lamin A/C levels were not changed in either WT or HSF4
del42 lenses when treated with rapamycin (
Fig. 3A). Then, we assessed the effect of rapamycin treatment on organelle degradation in HSF4
del42 lens in vivo. Newborn HSF4
del42 mice were injected intraperitoneally (I.P.) with 7.5 mg/kg rapamycin at p3, and then the lenses were dissected carefully for further analysis at p5. Our immunoblotting results indicated that I.P. administration of rapamycin efficiently increased LC3-II levels and reduced p62 levels in HSF4
del42 lens fibers as a consequence of autophagy induction (
Fig. 3B). Intraperitoneal injection of rapamycin caused sharp declines in GM130, GRP78, and Tomm20 protein levels in the lens fibers of HSF4
del42 mice compared with those of vehicle-injected mice (
Fig. 3B). These observations supported the idea that autophagy induction by rapamycin facilitated the degradation of detained membrane-bound organelles, such as mitochondria, endoplasmic reticulum, and Golgi apparatus, in HSF4
del42 lens fibers.