Fig. 2.

Hspa1a mRNA in ALS culture models using single molecule fluorescence in situ hybridization (smFISH). Cultures containing motor neurons expressing TDP-43^G348C, FUS^R521G, or SOD1^G93A were treated with vehicle (DMSO), 4 µM arimoclomol, 1 µM RGFP963, or the combination of arimoclomol and RGFP963 (Combo). smFISH was conducted on day 3. (a) The highly expressed Hspa8 mRNA is presented as a reference control for comparison to the minimal labeling of Hspa1a mRNA in (b)–(d). (b) No significant impact of TDP-43^G348C or drug treatments on the number of Hspa1a mRNA spots. (c) Scarcity of Hspa1a mRNA spots in neurons expressing FUS^R521G; no significant effect of drug treatments. (d) Low numbers of Hspa1a mRNA spots in neurons expressing SOD1^G93A; a small but significant increase in a subset of neurons by arimoclomol and RGFP963 treatments. Data are presented as mean ± SD, n = 9–24 neurons per group. (e–g) Number of transcription sites by smFISH. (e) Example of Hspa1a transcription site labeling. (f and g) Effect of ALS variants and combination drug treatment on the number of Hspa1a transcription sites on (f) day 1 and (g) day 3 following microinjection of expression vectors. n = 6–30 neurons per group. Statistical significance was evaluated through one-way ANOVA followed by Bonferroni post hoc analysis. *P < 0.05. Scale bar = 15 μm. Abbreviations used: ALS, amyotrophic lateral sclerosis; FUS, fused in sarcoma; SD, standard deviation; SOD1, superoxide dismutase I; TDP-43, TAR DNA binding protein 43 kDa. DMSO; dimethyl sulfoxide.