vcp inhibitor cb5083  (MedChemExpress)

Journal: Nature Communications

Article Title: DAF-16/FOXO and HLH-30/TFEB comprise a cooperative regulatory axis controlling tubular lysosome induction in C. elegans

doi: 10.1038/s41467-025-64832-x

Figure Lengend Snippet: a Endogenously tagged SPIN-1::mCherry in starved L1 worms with and without dSVIP overexpression. Scale bar, 5 µm. b Quantification of lysosomal junctions per object for the genotypes indicated. WT ( N = 20 worms), WT + dSVIP , daf-16(mu86) , daf-16(mu86) + dSVIP , hlh-30(tm1978) and hlh-30(tm1978) + dSVIP ( N = 30 worms per genotype) , daf-16(mu86); hlh-30(tm1978) and daf-16(mu86); hlh-30(tm1978) + dSVIP ( N = 20 worms per genotype). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (ns = not significant, ** p < 0.01, **** p < 0.0001) ( c ) Endogenously tagged SPIN-1::mCherry in fed day 1 adult worms with and without dSVIP overexpression. Scale bar, 5 µm. d Quantification of lysosomal junctions per object for the genotypes indicated. WT ( N = 28 worms), WT + dSVIP ( N = 20 worms), daf-16(mu86) ( N = 28 worms), daf-16(mu86) + dSVIP ( N = 28 worms), hlh-30(tm1978) ( N = 29 worms), hlh-30(tm1978) + dSVIP ( N = 25 worms) , daf-16(mu86); hlh-30(tm1978) ( N = 28 worms), daf-16(mu86); hlh-30(tm1978) + dSVIP ( N = 29 worms). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (ns = not significant, **** p < 0.0001). e Endogenously tagged SPIN-1::mCherry in fed WT ( N2 ) and daf-16(mu86) worms with gut dSVIP OE at day 1 of adulthood that were fed control DMSO or CB5083 VCP inhibitor beginning at L4 larval stage. Scale bar, 5 µm. f Quantification of lysosomal junctions per object for the genotypes and conditions indicated. WT ( N2 ) + DMSO ( N = 19 worms), daf-16(mu86); gut dSVIP OE + DMSO ( N = 18 worms), WT ( N2 ) + CB5083 ( N = 15 worms), daf-16(mu86); gut dSVIP OE + CB5083 ( N = 15 worms). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (**** p < 0.0001). g Endogenously tagged SPIN-1::mCherry in day 7 WT ( N2 ) animals that were fed DMSO or CB5083 VCP inhibitor beginning at day 5 of adulthood. Scale bar, 5 µm. h Quantification of lysosomal junctions per object. ( N = 20 control worms and N = 16 fed CB5083 worms). Mean ± s.e.m. Unpaired two-tailed Student’s t -test. (ns = not significant). i Endogenously tagged SPIN-1::mCherry in day 1 eat-2(ad1116) adults that were fed DMSO or CB5083 VCP inhibitor beginning at L4 larval stage. Scale bar, 5 µm. j Quantification of lysosomal junctions per object. eat-2(ad1116) +DMSO ( N = 20 worms), eat-2(ad1116) +CB5083 ( N = 16 worms). Mean ± s.e.m. Unpaired two-tailed Student’s t -test. (ns = not significant). For all experiments, data were pooled from two independent experiments.

Article Snippet: A 10 μM stock solution of the VCP inhibitor CB5083 (MedChem Express, Cat. # HY-12861/CS-5405) was prepared in DMSO and diluted to a final working concentration of 1 μM in M9 buffer.

Techniques: Over Expression, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: Image-based screen capturing misfolding status of Niemann-Pick type C1 identifies potential candidates for chaperone drugs

doi: 10.1371/journal.pone.0243746

Figure Lengend Snippet: (A) High-resolution clear native PAGE (fluorescence-detectable native PAGE) analysis of NPC1-GFP from cells treated as indicated. Cells stably expressing the indicated NPC1-GFP were treated with vehicle, mo56HC (3 μM), itraconazole (3 μM), or lapatinib (10 μM) for 21 h, and DDM-solubilized lysates were subjected to native-PAGE analysis after normalization with respect to total protein concentration. The native-PAGE gel was imaged for GFP fluorescence (upper panel), and total protein was also visualized by TCE staining (lower panel). (B) Ubiquitination status of NPC1-GFPs upon treatment with itraconazole or lapatinib. Cells stably expressing the indicated NPC1-GFP were treated as shown for 18 h followed by treatment with CB5083 (3 μM) for 6 h. The ubiquitination status was first probed with anti-Ub antibody after immunoprecipitation of the FLAG-NPC1-GFP, and then the amount of the FLAG-NPC1-GFP was re-probed with anti-FLAG antibody. The extent of ubiquitination was quantified and normalized to the amount of detected FLAG-NPC1-GFP. The raw, uncropped blot images are available from Mendeley Data repository ( http://dx.doi.org/10.17632/jr23ccpp46.3 ). (C) Quantification of the high-resolution clear native PAGE analysis from three independent experiments. The filled circles, error bars, and open circles represent mean, SEM, and raw data points from three independent experiments. Statistical significance was assessed by ANOVA and Tukey-Kramer multiple comparison test (***, p<0.001). The raw, uncropped gel images are available from Mendeley Data repository ( http://dx.doi.org/10.17632/jr23ccpp46.3 ). (D) Quantification of the ubiquitination assay data collected from three independent experiments. Data was normalized with the ubiquitination level of WT for each experiment. The filled circles, error bars, and open circles represent mean, SEM, and raw data points. Statistical significance of the treatment was assessed similarly as in (C). *, p<0.05; p<0.1; ns, not significant (p = 0.24).

Article Snippet: Chemical reagents used in this study and their commercial sources were as follows: VCP inhibitor CB5083 (Cayman), n-dodecyl-beta-D-maltoside (DDM, Dojindo), itraconazole (TCI), posaconazole (TCI), ketoconazole (TCI), ravuconazole (TCI), terconazole (Sigma-Aldrich), miconazole nitrate (TCI), hydroxyitraconazole (Toronto Research Chemicals), imatinib (TCI), lapatinib (Cayman Chemical), digoxin (TCI), progesterone (TCI).

Techniques: Clear Native PAGE, Fluorescence, Stable Transfection, Expressing, Protein Concentration, Staining, Ubiquitin Proteomics, Immunoprecipitation, Comparison