Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HIV-1 Vpr counteracts HLTF-mediated restriction of HIV-1 infection in T cells

doi: 10.1073/pnas.1818401116

Figure Lengend Snippet: HIV-1 Vpr antagonizes HLTF-mediated restriction of HIV-1 replication in T cells. ( A ) HLTF restricts HIV-1 replication. CEM.SS T cells harboring doxycycline-inducible codon-optimized HLTF transgene (CEM.SS_iHLTFo) were subjected to NT or targeting endogenous HLTF mRNA (HLTF) RNAi in the absence or presence of the indicated concentrations of doxycycline (ng/mL). ( Left ) Three days after initiation of RNAi, PRCA was performed with a 1:1 mixture of HIV-1.mRFP. vpr . wt and HIV-1.RFP. vpr . Q8 *. ( Right ) As a control, PRCA was also performed in parental CEM.SS T cells cultured in the absence or presence of doxycycline. ( Upper ) Cell-associated DNA of the competing viruses was quantified at 7 dpi. ( Middle ) Fold-change in HIV-1 DNA copies in cells subjected to HLTF RNAi, normalized to those in cells subjected to NT RNAi. ( Lower ) Percentages of cell-associated HIV-1 DNA for the competing viruses. ( B ) HLTF levels in CEM.SS_iHLTFo and parental CEM.SS T cell populations used in experiments shown in A were revealed by immunoblotting. Lamin B1 provided a loading control. ( C ) HLTF depletion partially restores the HIV-1 replication fitness advantage provided by Vpr. CEM.SS T cells were subjected to NT or HLTF-targeting (HLTF) RNAi and infected with a 1:1 mixture of HIV-1.mRFP. vpr . wt and HIV-1.RFP. vpr . Q8 *. Percentages of cell-associated DNA for the competing viruses at 7 dpi ( Left ) and fold-change in HIV-1 DNA copies per cell in cells subjected to HLTF-targeting RNAi normalized to those in cells subjected to NT RNAi ( Right ) are shown. The data shown are from five biological replicate experiments. *** P < 0.001.

Article Snippet: RNAi-resistant HLTF gene variants harboring HIRAN (HLTFo_HIRAN) or RING domain (HLTFo_RING) mutations were transduced into CEM.SS T cells as doxycycline-inducible transgenes.

Techniques: Control, Cell Culture, Western Blot, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HIV-1 Vpr counteracts HLTF-mediated restriction of HIV-1 infection in T cells

doi: 10.1073/pnas.1818401116

Figure Lengend Snippet: Exo1 and HLTF epistasis studies. ( A ) CEM.SS T cells were subjected to NT, Exo1-targeting, and/or HLTF-targeting RNAi, as indicated. PRCA with a 1:1 mixture of HIV-1.mRFP. vpr . wt and HIV-1.RFP. vpr . Q8 * was initiated 3 d later, and cell-associated DNA for each of the competing viruses was quantified at 7 dpi. Fold-change ( Left ) and relative percentages ( Right ) of cell-associated HIV-1 DNA copies per cell for the competing viruses are shown at 7 dpi. A representative of three experiments is shown. Only P values for HIV-1.RFP. vpr . Q8 * lower than 0.05 are shown: * P < 0.05; ** P < 0.01. ( B ) Exo1 and HLTF levels in extracts from cells subjected to RNAi assessed at the time of HIV-1 infection were revealed by immunoblotting. Lamin B1 provided a loading control.

Article Snippet: RNAi-resistant HLTF gene variants harboring HIRAN (HLTFo_HIRAN) or RING domain (HLTFo_RING) mutations were transduced into CEM.SS T cells as doxycycline-inducible transgenes.

Techniques: Infection, Western Blot, Control

Journal: Nature Communications

Article Title: FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops

doi: 10.1038/s41467-019-10179-z

Figure Lengend Snippet: FANCM supports normal cell-cycle progression and proliferation of ALT cells. a Western blot analysis of FANCM protein levels in ALT and Tel+ cells transfected with anti-FANCM siRNAs (siFa and siFb) or with control siRNAs (siCt). ALT cells (gray background) are: U2OS, HuO9, Saos2 and WI-38 VA13 (VA13); Tel+ cells are: HeLa, HOS, HT1080 (HT10) and SKNAS (SK). U-ST and H-ST are supertelomerase U2OS and HeLa cells, respectively. Proteins were extracted 48 h after transfection. Lamin B1 (LMB1), Golgin 97 and KAP1 serve as loading controls. b Examples of FACS profiles of the indicated siRNA-transfected cells stained with propidium iodide (PI). Cell counts ( y axis) are plotted against PI intensity ( x axis). Cells were harvested 48 h after transfection. c Quantifications of experiments as in ( b ). The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. d Examples of colony formation assays with the indicated siRNA-transfected cells. e Quantifications of experiments as in ( d ). The graph shows colony numbers relative to siCt-transfected samples. Bars and error bars are means and SDs from three independent experiments. P values were calculated with a two-tailed Student’s t test. * P < 0.05, ** P < 0.005, *** P < 0.001. f Growth curves of U2OS and HeLa cells transfected with the indicated siRNAs every 3 days. Cell numbers are expressed relative to siCt-transfected cells. Data points and error bars are means and SDs from three independent experiments. g Western blot analysis of U2OS cells infected with retroviruses expressing Flag-tagged TRF1 (FL-TRF1) or with empty vector (ev) control retroviruses. Five days after infections cells were transfected with the indicated siRNAs and harvested 48 h later. pS33: RPA32 phosphorylated at serine 33, pRPA32: phosphorylated RPA32. LMB1 and Golgin serve as loading controls. h Examples of FACS profiles of cells as in ( g ). The graph on the left shows the percentage of cells in G1, S and G2/M phases from one representative experiment. Source data are provided as a Source Data file

Article Snippet: For FANCM complementation experiments, siFa- and siFb-resistant cDNAs coding for N-terminally V5-tagged FANCM variants were synthesized at GenScript and cloned into the into the lentiviral vector pLVX-TetOne-Puro (Clontech).

Techniques: Western Blot, Transfection, Staining, Two Tailed Test, Infection, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops

doi: 10.1038/s41467-019-10179-z

Figure Lengend Snippet: BLM depletion substantially averts the phenotypes associated with FANCM depletion. a Western blot analysis of FANCM and BLM in U2OS cells transfected with siFa, anti-BLM siRNAs (siBl), and siCt. Two different concentrations (5 and 20 nM) of siFa were used. Cells were harvested 48 h after transfection. LMB1 serves as loading control. The asterisk indicates a band cross-reacting with the anti-FANCM antibody. b Quantifications of FACS profiles of cells as in ( a ) stained with PI. The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. c Example of colony formation assays using cells as in ( a ). siFa5: 5 nM siRNA, siFa20: 20 nM siRNA. The graph on the right shows colony numbers relative to siCt-transfected samples. Bars and error bars are means and SDs from four independent experiments. P values were calculated with a two-way ANOVA followed by Tukey’s HSD. d Growth curves of U2OS cells transfected with the indicated siRNAs (20 nM each) every 3 days. Cell numbers are expressed relative to siCt-transfected cells. Data points and error bars are means and SDs from three independent experiments. SiCt and siFa curves are the same as the ones shown in Fig. . e Examples of pS33 immunostaining (red) combined with TRF2 immunostaining (green) on cells as in ( a ). In the merge panel, DAPI-stained DNA is also shown (blue). Arrowheads point to pS33 TIFs. Scale bar: 10 μm. f Quantifications of numbers of pS33 TIFs per nucleus in cells as in ( a ). Each dot represents an individual nucleus. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. P values were calculated with a two-way ANOVA followed by Tukey’s HSD. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file

Article Snippet: For FANCM complementation experiments, siFa- and siFb-resistant cDNAs coding for N-terminally V5-tagged FANCM variants were synthesized at GenScript and cloned into the into the lentiviral vector pLVX-TetOne-Puro (Clontech).

Techniques: Western Blot, Transfection, Staining, Immunostaining

Journal: Nature Communications

Article Title: FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops

doi: 10.1038/s41467-019-10179-z

Figure Lengend Snippet: Deregulated telR-loops contribute to the replication stress arising upon FANCM depletion in ALT cells. a Western blot analysis of U2OS cells infected with retroviruses expressing V5 epitope-tagged FANCM variants or empty vector (ev) control retroviruses. WT wild type, K117R ATPase/translocase dead FANCM. Five days after infections cells were transfected with the indicated siRNAs and harvested 48 h later. LMB1 serves as loading control. b Quantifications of FACS profiles of cells as in ( a ) stained with PI. The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. c Quantifications of numbers of pS33 TIFs and APBs per nucleus in cells as in ( a ). Each dot represents an individual nucleus. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. P values were calculated with a two-way ANOVA followed by Tukey’s HSD. d Western blot analysis of U2OS cells infected with retroviruses expressing MYC epitope-tagged RNaseH1 (RH1) variants or ev control retroviruses. D145A: endoribonuclease dead RNaseH1. Five days after infections cells were transfected with the indicated siRNAs and harvested 48 h later. siF/B: combined siFa and siBl. LMB1 serves as loading control. e Quantifications of FACS profiles of cells as in ( d ) stained with PI. The graph shows the percentage of cells in G1, S and G2/M phases from one representative experiment. f Quantifications of numbers of pS33 TIFs per nucleus in cells as in ( d ). Each dot represents an individual nucleus. A total of at least 300 nuclei from three independent experiments were analyzed for each sample. Bars and error bars are means and SDs. P values were calculated with a two-way ANOVA followed by Tukey’s HSD. Comparisons between D145A and ev and WT are not indicated. * P < 0.05, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file

Article Snippet: For FANCM complementation experiments, siFa- and siFb-resistant cDNAs coding for N-terminally V5-tagged FANCM variants were synthesized at GenScript and cloned into the into the lentiviral vector pLVX-TetOne-Puro (Clontech).

Techniques: Western Blot, Infection, Expressing, Plasmid Preparation, Transfection, Staining