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. 2021 Mar 5;12:1484. doi: 10.1038/s41467-021-21611-8

Fig. 6. Bidirectional regulation between VHP-1, KGB-1, and JNK signaling influences neurodegeneration following mechanical stress.

Fig. 6

a Western blot of phosphorylated (P-) KGB-1, p38, and ERK in response to injury and/or vhp-1 RNAi. Tubulin, loading control. b Quantification of phospho-specific western blots relative to uninjured empty vector. Mean + SEM. From left to right, ns (not significant), *p = 0.0116, *p = 0.0109, **p = 0.0031, **p = 0.0049 by two-way ANOVA with Tukey test. n = 3 biological replicates. KGB-1 (black), p38 (gray), ERK1/2 (stripes). c, d qPCR of lys-3 or vhp-1 transcript abundance either uninjured (black) or 1 h post injury (blue) in wild type or kgb-1(um3) mutants with the respective RNAi. Mean ± SEM. ****p < 0.0001, ***p = 0.0004 (EV) and ***p = 0.0002 (fos-1 RNAi) by two-way ANOVA with Tukey test. n = 3 biological replicates. e Confocal micrographs of JUN-1::GFP expression in the worm head with and without vhp-1 RNAi and injury (4 h post trauma). Differential interference contrast, DIC. Reference Fig. 6f. Scale = 50 µm. f Quantification of JUN-1::GFP by large-particle flow cytometry comparing fluorescence in the worm head for uninjured (Uninj, gray) and 4 h post injury (Inj, blue) with and without vhp-1 RNAi. Mean ± SEM. Two-way ANOVA with Tukey multiple comparison test. See Supplementary Data 5 for statistics. g JUN-1 transcript abundance determined by RNAseq without injury (black) and 1 h post injury (blue). Mean ±  SEM. **p = 0.003 by two-sided unpaired t-test. n = 3 biological replicates. Reads per kilobase million (RPKM). h, i Large-particle flow cytometry showing the effects of trauma on dopaminergic GFP retention 48 h post injury. Shown are values indexed to empty vector control (EV). Mean ± SEM. h vhp-1 RNAi in wild type (green) or kgb-1(um3) mutants (black). i effects of jun-1 RNAi with vhp-1 RNAi (black) compared to vhp-1 RNAi alone (green). Positive values indicate reduced loss of fluorescence when compared to age-matched uninjured controls. A value of one indicates no change of fluorescence compared to uninjured controls. See Supplementary Data 5 for statistics.