Fig 2: hsc70-4 downregulation reduces the aggregates accumulation, resulting in the improved arrangement of rhabdomeres and optic neurons. Thus, it enhances the phototaxis response. (A) The panel shows the PolyQ aggregates (in red) accumulation in (a) GMR-GAL4:UAS-127Q; (b) GMR-GAL4:UAS-127Q>UAS-Hsc70-4RNAi; (c) GMR-GAL4:UAS-127Q> UAS-Hsc70-4WT. Panels (d–f) from the panel (A) show the nuclei staining (in blue) along with the aggregates of the respective genotypes (scale bar 20 µm). (B) Quantitative analysis of the intensity/pixel of the immunofluorescence images shows PolyQ aggregates accumulation in the different genotypes from three independent experiments and data shown as ±SEM (ns indicates insignificant change at p-value = 0.05%, and * indicates the significance at a p-value of = 0.05). (C) hsc70-4 downregulation restores the rhabdomere arrangement and the axonal connection disruption in GMR-GAL4:UAS-127Q-driven flies. (a,e) Oregon-R+, (b,f) GMR-GAL4:UAS-127Q, (c,g) GMR-GAL4:UAS-127Q> UAS-Hsc70-4RNAi, (d,h) GMR-GAL4:UAS-127Q> UAS-Hsc70-4WT. Anti-ELAV (in green) to mark rhabdomere and 22C10 staining against Futsch (in green) to visualize the optic neurons (scale bar 20 µm). (D) The graph shows the phototaxis response in the 10-day-old flies of Oregon-R+, GMR-GAL4:UAS-127Q, GMR-GAL4:UAS-127Q> UAS-Hsc70-4RNAi, and GMR-GAL4:UAS-127Q> UAS-Hsc70-4WT (the experiment was performed in triplicate where n =100 for each genotype, and data are shown as ±SEM; * indicated significance at the p-value of =0.05%).

Fig 3: Alterations in the hsc70 gene isoforms in the polyQ-overexpressing eye phenotype. (A) Transcript expressions of hsc70-1, hsc70-3, and hsc70-4 are upregulated compared to hsc70-2 and hsc70-5, while that of hsf is reduced in GMR-GAL4:UAS-127Q in comparison to Oregon-R+(normalization is done using rp49). (B) Western blot showing the expression of Hsc70 in different genotypes. (C) Graph shows quantitative analysis of Hsc70 expression normalized to beta-tubulin, showing significant upregulation of the protein in lane 2 and 4 compared to lane 1. In contrast, the protein level of Hsc70 in the lane 3 was comparable to lane 1. However, it was significantly downregulated compared to lane 2 (n = 3 and data shown as ±SEM and * indicated significance at the p-value of =0.05). (D) Eye degeneration in GMR-GAL4:UAS-127Q background is rescued after the downregulation of hsc70-4. Normal adult eyes in Oregon-R+ (a–d), disrupted in GMR-GAL4:UAS-127Q flies (e–h), as they show distorted ommatidial pattern along with the loss of eye pigmentation due to expanded PolyQ aggregates accumulation. Downregulating hsc70-4 restores the ommatidial arrangement and eye pigmentation in polyQ-driven flies (i–l), while overexpressing hsc70-4 does not correct the ommatidial arrangement and eye pigmentation in polyQ-driven flies (m–p). The nail polish imprint of adult eyes showing the ommatidia pattern is in the inset of each image in this panel (scale bar 100 µm).

Fig 4: Elevated immune responses in the polyQ background get reduced upon hsc70-4 downregulation. (A) The histogram shows qPCR analysis representing the fold change in transcript level of relish and AMPs in GMR-GAL4:UAS-127Q conditions significantly elevated in comparison to the wild-type. But GMR-GAL4:UAS-127Q> UAS-Hsc70-4RNAi shows a significant decline in these respective immune genes (data normalized to rp49, three biological replicated were used, and data are shown as ±SEM; ns means insignificant change at p-value of =0.05, and *indicated significance at the p-value of =0.05). (B) Reduction in expression of Relish (green) and HSP70 (red) after downregulation of hsc70-4 in GMR-GAL4:UAS-127Q>UAS-Hsc70-4RNAi in comparison to GMR-GAL4:UAS-127Q, in lower magnification (a–d,f–i,k–n) and (e,j,o) shows the higher magnification (scale bar 20 µm). The inset in (e,j,o) was captured at a further 3X zoom to show co-localization (scale bar 10 µm). Genotype description, Oregon-R+ (a–e); GMR-GAL4:UAS-127Q (f–j); GMR-GAL4:UAS-127Q> UAS-Hsc70-4RNAi (k–o).

Fig 5: PolyQ aggregates colocalized and coprecipitated with Hsc70 and Relish. (A) The panel shows the section image of the eye imaginal disc of GMR-GAL4 UAS 127Q third instar larvae. (a–d) Co-staining of the PolyQ aggregates (in red) and Hsc70 (in green) shows that PolyQ aggregates and Hsc70 colocalize with each other; the arrow demarcates the colocalized regions in yellow (scale bar 5 µm). (e–h) Co-staining of the PolyQ aggregates (in red) and Relish (in green) shows that PolyQ aggregates colocalize with Relish. Arrow demarcates the colocalized regions in yellow (scale bar 5 µm). (i–l) Co-staining of Hsc70 (in red) and Relish (in green) co-stained in the polyQ-expressing eye disc shows the colocalization (in yellow), which is marked by the arrow (scale bar 5 µm). (B) Immunoblot panels illustrated the presence of the interacting proteins in the co-IP elute (denoted as IP) from the crude protein sample (demarcated as IN, means input) of GMR-GAL4:UAS-127Q.HA background. (i) Western blot shows the presence of Relish and Hsc70 in the IP1 sample after precipitation of PolyQ aggregate using anti-HA and the expression pattern in input. Images ii and iii are immunoblot showing the presence of Hsc70 and PolyQ aggregates, respectively, in the IP2 sample after precipitation of Relish using anti-Relish, and the expression pattern was compared with the input sample. IP 1—eluted protein sample after pull-down with anti-HA; IP 2—eluted protein sample after pull-down with anti-Relish; IN—crude protein sample of GMR-GAL4:UAS-127Q.HA genetic background; 1- whole-Relish protein (~110 kDa), 1* donates a faint band of N-terminal Relish (~68 kDa), and 1# denotes a sharp band of C-terminal Relish (~49 kDa); 2—Hsc70 (~71 kDa); 3—PolyQ aggregates oligomers detected by anti-HA along with *representing the bands of anti-HA; H and L denote heavy chain (~55 kDa) and light chain (~25 kDa) of IgG.