Fig 1: Vpr promotes depletion of CTIP2 in primary CD4+ T cells and at the HIV-1 promoter to induce viral reactivation in microglial cells. (A) Relative CTIP2 expression in CD4 T cells was quantified by flow cytometry. Expression levels in cells incubated with Virus Like-Particles VLP-HA-Vpr were compared to the control VLP-HA particles taken as 1. (B) CHME5-HIV latently infected microglial cell line was infected with VPLs or treated by TNFa. The number of GFP+ cells was quantified by flow cytometry and presented relative to GFP+ cells obtained after VLP-HA infections. (C) Chromatin Immuno-precipitation experiments targeting endogenous CTIP2 at the HIV-1 promoter were performed with chromatin from microglial cells expressing the chromatinized LTR-LUC episomal vector and Vpr as indicated. The cells were subjected or not to MG132 treatment. Enrichments are presented as percentages of the inputs. As a control, the presence of CTIP2 has been concomitantly quantified at the luciferase gene region. Results are representative of at least three independent experiments.
Fig 2: Branaplam reduces total and mutant HTT protein levels in a dose-dependent manner without inducing toxicity.a Paradigm: cell types and parameters analyzed. Created with BioRender.com. b–d tHTT levels (2B7/D7F7 assay), mHTT levels (2B7/MW1 assay) and toxicity (adenylate kinase release, Triton: positive control) in fibroblasts (b), iPSC (c) and cortical progenitors (d) with different Branaplam concentrations for 72 h. Green: Ctrl samples (n = 2 33Q/33Q-S1-109, 4L6/4L6-S1-027), purple: HD samples (n = 2 919/919-S1-101, 4Q4/4Q4-S1-109). b Fibroblasts statistics: tHTT: one-way ANOVA with Geisser–Greenhouse correction (P = 0.0012); mHTT: no statistics applied; toxicity (Triton excluded): Friedman test (P = 0.0770). c iPSC statistics: tHTT one-way ANOVA with Geisser–Greenhouse correction (P = 0.0032); mHTT: no statistics applied; toxicity (Triton excluded): one-way ANOVA with Geisser–Greenhouse correction (P = 0.0455), no significant differences in multiple comparisons. d cortical progenitor statistics: tHTT IC50: 919-S1-101 = 2.233 × 10-9 M; 4Q4-S1-109 = 6.102 × 10-9 M; 33Q-S1-109 = 3.191 × 10-9 M; 4L6-S1-027 = 1.182 × 10-9 M; mHTT: 919-S1-101 = 5.528 × 10-9 M; 4Q4-S1-109 = 8.952 × 10-9 M; 33Q-S1-109 and 4L6-S1-027 = not calculated; toxicity (Triton excluded): one-way ANOVA with Geisser–Greenhouse correction (P = 0.06). e tHTT levels (2B7/D7F7 assay), mHTT levels (2B7/MW1 assay) (Ctrl n = 3; HD n = 4) and toxicity (adenylate kinase release, Triton as positive control) in cortical neurons of (Ctrl n = 4; HD n = 4). tHTT and mHTT measured with DMSO (dark shades) or 10 nM Branaplam (light shades) for 72 h. Statistics: tHTT: 2-way ANOVA (DMSO vs. Branaplam: P = 0.0009; Ctrl vs. HD: P = 0.6820; interaction: P = 0.3833); mHTT: two-way ANOVA (DMSO vs. Branaplam: P = 0.0614; Ctrl vs. HD: P = 0.0223; interaction: P = 0.0615); toxicity (Triton excluded): Friedman test (P = 0.5306). Bars: median ± IQR. f Bar plot showing number of Casp3/7 positive beta-III-Tubulin+ and CTIP2+ cortical neurons after DMSO (dark shades) or (light shades) 72 h 10 nM Branaplam treatment (Ctrl n = 4; HD n = 4). Statistics: beta-III-Tubulin+: two-way ANOVA (DMSO vs. Branaplam: P = 0.0782; Ctrl vs. HD: P = 0.4744; interaction: P = 0.9502); CTIP2+: 2-way ANOVA (DMSO vs. Branaplam: P = 0.7230; Ctrl vs. HD: P = 0.5779; interaction: P = 0.6348). Bars: median ± IQR. Source data are provided as a Source Data file.
Fig 3: Mutant HTT is increased in HD patient-derived cells using an MSD assay.a Paradigm illustrating the HD patient-based disease model (fibroblasts, iPSC, cortical progenitors (25d old), and cortical neurons (35d old)) and readouts. Created with BioRender.com. b Bar plot depicting FACS quantification of NESTIN/PAX6 double-positive cells. Statistics: Welch’s test. Bars: median ± IQR. c Bar plot illustrating FACS quantification of bIII-Tubulin/CTIP2 double-positive cells. Statistics: Welch’s test. Bars: median ± IQR. d Representative pictures of cortical neurons. Scale bar 50 µm. e Illustration depicting the MSD HTT quantification assay, where the added protein samples bind to 2B7 antibody, used for coating the plates. The SULFO-TAG coupled antibodies D7F7 and MW1 are added for quantification of total HTT and mutant HTT, respectively. Note: numeric values from 2B7/D7F7 assay (total HTT) cannot be directly set in relation to numeric values from 2B7/MW1 assay (mutant HTT). Created with BioRender.com. f Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in fibroblasts (4 Ctrl lines, 4 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test (P value = 0.9717); mHTT Welch’s test (P value = 0.0737). Bars: median ± IQR. g Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in iPSC (8 Ctrl lines, 9 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test (P value = 0.2080); mHTT Mann–Whitney test (P value < 0.0001). Bars: median ± IQR. h Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in cortical progenitors (7 Ctrl lines, 9 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test (P value = 0.2149); mHTT Welch’s test (P value = 0.0061). Bars: median ± IQR. i Bar plots quantifying total (tHTT, top) and mutant (mHTT, bottom) levels in cortical neurons (7 Ctrl lines, 8 HD lines) with 2B7/D7F7 and 2B7/MW1 MSD assays, respectively. Statistics: tHTT: Welch’s test (P value = 0.2781); mHTT Welch’s test (P value = 0.0239). Bars: median ± IQR. Source data are provided as a Source Data file.
Fig 4: CTIP2-induced expression in infected cells is counteracted by HIV-1 in a Vpr dependent manner. (A,C) Jurkat T cells (A) and Microglial cells (C) were infected with 25 × 103 copies/ml of a VSV-G pseudotyped NL4.3?Env-LUC provirus for 24 h. The presence of CTIP2 in infected cells was assessed by western blot and the CTIP2 mRNA quantified by RT-Q-PCR. (B,D) Jurkat T cells (B) and Microglial cells (D) were infected with 150 × 103 copies/ml of a VSV-G pseudotyped NL4.3?Env-LUC (WT) and a NL4.3?Env?Vpr -LUC (?Vpr) provirus for 24, 48 and 96 h. The presence of CTIP2 in infected cells was assessed by western blot and the viral expression was quantified by luciferase assay. The quantifications are presented relative to the quantities obtained after 24 h of infection. The results are representative of at least three independent experiments.
Fig 5: DCAF1 binding is necessary for Vpr-mediated degradation of CTIP2. (A) Nuclear extracts from control or DCAF1 knock-down cells were analyzed by western blot for the presence of the indicated proteins in the presence of GFP-Vpr and the control GFP. CTIP2 expression was quantified relative to the loading control using ImageJ software. DCAF1 knock-down efficiency in our experiments is presented. (B) Nuclear extracts from cells expressing the indicated proteins were analyzed by western blot. ß-actin is presented as a loading control. (C) Nuclear extracts from cells expressing large amount of Tap-CTIP2 (t-CTIP2) and the indicated HA-Vpr proteins were subjected to immunoprecipitation experiments targeting the HA tag. The input and the Vpr-associated complexes were analyzed for the presence of CTIP2 by western blot.
Supplier Page from Abcam for Anti-Ctip2 antibody [25B6] (FITC)