Taken together, these data provide strong evidence that Tat levels are regulated by selective autophagy through a K63Ub-dependent mechanism. Selective protein destruction can occur either due to canonical macroautophagy or due to chaperone-mediated autophagy (CMA), or both, however the mechanisms of each pathway are quite unique [48, 49]. 0.5 h (pink collection), 2 h (yellow collection), and 6 h (light blue collection) before ChIP, respectively. ChIP antibodies were described in Methods.(TIF) ppat.1007071.s002.tif (433K) GUID:?F5F3251F-EF3A-4983-93AD-EDE24ED842B7 S3 Fig: JIB-04 has only minor effects on host cell transcription at low concentration (Related to Fig 3). (A) Pie-chart of the 811 genes (out of 13546 genes with reads 10) altered more than 2-fold by JIB-04 (p 0.05). These 811 genes were outlined in S1 File. (B) The top ten Gene Ontology enrichment biological process terms for DMSO vs JIB-04 and TNF 0 h vs 6 h. (C) qRT-PCR results for the indicated genes randomly-selected from the top 100 heatmap for histones and Rabbit polyclonal to OSBPL6 JIB-04 activated genes, respectively. The significant differences between DMSO-treated and JIB-04-treated samples were analyzed by Students T-test (*** = p 0.0005). (D) Immunoblot analysis of histone H2B and H3 protein levels in 2D10 cells that were exposed to JIB-04 (0C10 M) for 24 h. Csn3 served as loading control.(TIF) ppat.1007071.s003.tif (1.2M) GUID:?64FA8A82-7C7B-4765-93ED-A0A072DA22AA S4 Fig: JIB-04 inhibited HIV replication with high cell toxicity in main CD4+ T cells (Related to Fig 4). Graph show the data of analyzing JIB-04 in main CD4+ T cells. The percentage of intracellular HIV-p24 was used VU 0238429 to monitor the inhibition effect of the compounds. No treatment with HIV contamination sample was set as unfavorable control. DMSO plus 500 nM of commercial HIV-drug Raltegravir-treatment sample was set as positive control. The inhibition% values of the Y-axis were calculated by the formula (inhibition% = (p24% of no treatmentCp24% of the respective treatments) / p24% of no treatment*100%). Raltegravir treatment reached 100% inhibition so as high concentrations of JIB-04. The unfavorable value of DMSO-treatment showed DMSO treatment promoted contamination. The viability of main T cells was shown by the orange collection.(TIF) ppat.1007071.s004.tif (525K) GUID:?E926DAC8-5391-4A28-B8CD-DC557331184B S5 Fig: Knockdown of various KDMs failed to recapitulate the effect of JIB-04 on Tat expression (Related to Fig 5). (A) One representative immunoblot for the indicated factors at the conditions of knocking down the JMJDs/KDMs in 2D10 cells (KDM4D, Csn3, USP7, Csn8 and Cyclin T1 as loading controls). (B) One representative immunoblot for the indicated factors at the conditions of knocking down KDM4C in Tet-on-Tat-off HeLa cells (Cyclin T1, loading control). (C) Schematic diagram of protocol for panel A in 2D10 cells.(TIF) ppat.1007071.s005.tif (1.0M) GUID:?AB903DFA-496F-4618-9D3A-4FD175BC940E S6 Fig: VU 0238429 JIB-04 increases proteolytic destruction of Tat protein (Related to Fig 6). (A) Titration of JIB-04 in Tet-on-Tat-off HeLa cells. Top, immunoblot for the inidcated proteins at the concentrations of JIB-04. Cyclin T1 served as loading control. Bottom, qRT-PCR for HA-Tat86 mRNA levels at the same concentrations of JIB-04 as in top penal. Tat mRNA was normalized to mRNA and Tat mRNA treated with Doxycycline was normalized to 1 1. (B) Top, Dual-Luc assay analysis for HIV-LTR-Luc at the indicated treatments in VU 0238429 Tet-on-Tat-off HeLa cells. HIV-LTR-Luc was normalized to SV40-Renilla-Luc. Middle, Luc assay analysis for SV40-Renilla-Luc at the same condition. Renilla-Luc activity was normalized to total protein concentrations. Bottom, CMV–Gal assay for CMV–Gal at the same condition. CMV–Gal activity was normalized to Renilla-Luc activity. Activity from cells treated by 10 g/ml doxycycline was normalized to 1 1. VU 0238429 The significant differences between luciferase and -Gal activity for DMSO and JIB-04 treated samples were calculated by Students T-test (ns = non-significant, *p 0.05). (C) Left, immunoblot results showed the half life of the indicated proteins in 2D10 T cells treated by 1 M cycloheximide (Chx) and pre-treated with DMSO or 5 M JIB-04 for 1 h. Cyclin T1 served as loading control. Right, relative levels of Tat was measured by Image J and graphed.(TIF) ppat.1007071.s006.tif (855K) GUID:?A1D88094-6BD3-4739-BEEC-B369967537A2.
