FIGURE 3 | Mod(mdg4) as a downstream target of ENT2/AdoR pathway modulated mHTT effects and aggregate formation. (A) Survival of mHTT-expressing adult females (elav > Q93) with RNAi co-silencing (Ri) transporters ( ent1 or ent2 ), and adoR . Significance was analyzed by a weighted log-rank test; significant differences between each treatment group and control ( gfp -Ri) are labeled as follows: *** P < 0.001; N.S., not significant. n > 200. (B,C) Microarray analysis of the transcriptomes of ent2 and adoR mutants. Venn diagram shows the number of common genes (in intersect region) which were upregulated (B) or downregulated (C) among the adoR mutant larvae vs. control (w 1118 ), adoR mutant adults vs. control (w 1118 ), and ent2 mutant larvae vs. control (w 1118 ). The cutoff values for expression differences were set at Q < 0.05 (false discovery rate, FDR). (D) Survival of mHTT-expressing adult females (elav > Q93) with RNAi co-silencing (Ri) of potential downstream genes of the ENT2/AdoR pathway. Significance was analyzed by a weighted log-rank test; significant differences between each treatment group to control ( adoR -Ri) are labeled as follows: *** P < 0.001; N.S., not significant. n > 200. (E) Retinal pigment cell degeneration in mHTT-expressing adult females (gmr > Q93) with RNAi silencing potential downstream genes of the ENT2/AdoR pathway. Blue arrows indicate treated groups showing a significantly reduced loss of pigment. Detailed methodologies for sample collection and eye imaging are described in section “Materials and Methods.” (F) Representative confocal images of the brains of 10-day-old mHTT-expressing adult females (elav > Q93) with RNAi silencing potential downstream genes of the ENT2/AdoR pathway. Neuronal cells were detected with anti-Elav and mHTT aggregates were detected with anti-HTT (MW8). (G) The level of mHTT aggregate formation was calculated by normalizing the area of mHTT signal to the area of Elav signal. Significance in mHTT aggregate levels was analyzed using a Mann–Whitney U -test; significant differences between control Q93 flies and each RNAi treatment group are labeled as follows: * P < 0.05; ** P < 0.01; N.S., not significant. Error bars are presented as mean ± SEM. The number (n) of examined brain images are indicated above each bar. (H) Survival of mHTT-expressing adult females (elav > Q93) with co-RNAi silencing mod(mdg4) and co-overexpressing adoR or ent2 . Significance was analyzed by a weighted log-rank test; significant differences are labeled as *** P < 0.001. n > 200. (I) Transcription level of mod(mdg4) 2 h after Ado injection into the whole body of 3- to 5-day old control adult females (elav-gal4/+) and adoR RNAi females (elav > adoR-Ri). Significance was analyzed by Student’s t -test and labeled as follows: ** P < 0.01; N.S., not significant. n = 3. Error bars are presented as mean ± SEM. (J) Transcription levels of mod(mdg4) in Q93-expressing (elav > Q93) and control Q20-expressing (elav > Q20) larval brains. Significance was analyzed by Student’s t -test and labeled as * P < 0.05. n = 3. Error bars are presented as mean ± SEM.