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[Preprint]. 2024 Oct 5:2023.10.03.560761. Originally published 2023 Oct 3. [Version 2] doi: 10.1101/2023.10.03.560761

Fig. 5. Localized HSP mRNA translation in primary neurons.

Fig. 5.

(a) Representative IF detection of HSPA1A in Ctrl, MG132-stressed (10 μM for 7 h), and MG132-recovered (10 μM MG132 for 7 h and washout for 8 h) primary spinal cord motor neurons. (b) The plot shows the quantification of HSPA1A in three independent experiments (n = 45 total neurons). ****; P < 0.0001, ***; P < 0.001, ns; no significant (by 1 way ANOVA). (c) Representative images of puro-PLA detection of newly synthesized HSPA8 peptides. Scale bar = 10 μm. (d) Quantification of the number of puro-PLA signals detected per area of soma and dendrite in three independent experiments (n = 51–109 neurons and n = 123–163 dendrites; dots indicate individual soma and dendrite values). **; P < 0.01, ns; no significant (by 1 way ANOVA). (e) Schematic of the Hspa8 single-molecule translational reporter and the IF-smFISH signals expected for untranslated mRNAs and those being translated by a monosome or polyribosomes. Distention among them was based on the intensity of the IF signal colocalizing with the mRNA, which is proportional to the number of nascent peptides produced from an mRNA. (f) Representative IF-smFISH image of dendrites of an MG132-stressed primary spinal cord motor neuron expressing the Hspa8 single-molecule translational reporter (e). The black arrowhead indicates the direction of the dendrite from the soma. Squares depict the magnified regions. White arrows indicate translating mRNAs. Scale bar = 10 μm. (g, i) Quantification of the percentage of Hspa8 and ActinB translated mRNAs per dendrite. Data are the mean ± SD of (g) seven independent experiments (n = 70–88 dendrites) and (i) three independent experiments (n = 58–74 dendrites). *, P < 0.05; ns, no significant (by unpaired t-test). (h, j) Quantification of the relative nascent peptides intensity (RPI) colocalizing with a translating Hspa8 and ActinB mRNAs from (g) and (i). Media and SEM of translating mRNAs. M = monosomes, LP = light polyribosomes, and HP = heavy polyribosomes.