Fig. 8: RAB3C and RAB12 are involved in BCH-HSP-C01-mediated vesicle trafficking and enhancement of autophagic flux.

a AP4B1^KO SH-SY5Y cells transfected for 72 h with RNPs targeting RAB3C, RAB12 or both compared to NLRP5 (non-essential control). Vehicle vs. BCH-HSP-C01 treatment at 5 µM was administered for 24 h. Data points represent per well means. Each experimental condition was tested in multiple replicates (n_{AP4B1KO + sgNLRP5}: 20 wells from 5 independent plates; n_{AP4B1KO + sgNLRP5 + BCH-HSP-C01}: 18 wells from 5 independent plates; n_{AP4B1KO + sgRAB3C}: 24 wells from 5 independent plates; n_{AP4B1KO + sgRAB3C + BCH-HSP-C01}: 22 wells from 5 independent plates; n_{AP4B1KO + sgRAB12}: 28 wells from 5 independent plates; n_{AP4B1KO + sgRAB12 + BCH-HSP-C01}: 25 wells from 5 independent plates; n_{AP4B1KO + sgRAB3C + sgRAB12}: 22 wells from 3 independent plates; n_{AP4B1KO + sgRAB3C + sgRAB12 + BCH-HSP-C01}: 22 wells from 3 independent plates). Statistical testing was done using the t-test. P-values are two-sided. b Representative images. Scale bar: 10 µm. c Representative western blots. Cells were treated with vehicle vs. BCH-HSP-C01 at 5 µM for 72 h. d–f Quantification of western blots. Experiments were performed in four biological replicates. Error bars represent ± 1 SD. Statistical testing was done using the t-test. P-values are two-sided and were adjusted for multiple testing using the Benjamini-Hochberg procedure. g, h AP4B1^KO SH-SY5Y cells treated with BCH-HSP-C01 (5 µM) were incubated with ascending non-toxic doses of bafilomycin A1 (5 nM or 10 nM) or chloroquine (1 µM or 2 µM) for 24 h. Each condition was tested in 16 wells from 2 independent plates. (AP4B1^KO). i Representative images. Scale bar: 10 µm. j–l Representative western blots and quantification of whole cell lysates of AP4B1^KO SH-SY5Y cells transfected for 72 h with RNPs against RAB3C, RAB12 or both, compared to NLRP5. Vehicle vs. BCH-HSP-C01 treatment was administered for 48 h. Error bars represent ± 1 SD. Statistical testing was done using the t-test. P-values are two-sided and were adjusted for multiple testing using the Benjamini-Hochberg procedure. Box plots in all experiments show medians (center), upper and lower quartiles (hinges) and 1.5 x IQR (whiskers). Dashed lines represent a reduction of the ATG9A ratio of −2 SD compared to negative controls.