Journal: PLOS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

doi: 10.1371/journal.ppat.1011080

Figure Lengend Snippet: A: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Conditioned media harvested from rKSHV.219 latently infected iSLK cells was used to mimic the KS lesion microenvironment and induce the transcription of cytokines for 6 h prior to lysis for total RNA (normal media was used for the 0 h time point). Transcript levels were quantified by qPCR and normalized to 18S as a reference gene. Data is represented as the fold change in target transcript expression relative to the untreated vector control and was quantified using the ΔΔCq method. Results were plotted in GraphPad, a 2-way ANOVA with a Tukey’s multiple comparisons test was performed, ±SEM; n = 3, * = P<0.05, ** = P<0.01. B: HUVECs were treated with Torin (250 nM) or a DMSO control for 2 h prior to lysis for total RNA. Transcript levels were quantified by qPCR and normalized to HPRT as a reference gene. Data is represented as the fold change in target transcript expression relative to the untreated vector control and was quantified using the ΔΔCq method. An unpaired t-test was performed, ±SEM; n = 3, * = P<0.05. C: HeLa Tet-Off cells were co-transfected with expression plasmids for an ARE-containing firefly luciferase plasmid (pTRE-FLuc-ARE) and a stable renilla luciferase plasmid (pTRE-RLuc). 36 h post transfection, doxycycline (Dox) was added to halt reporter gene transcription of both luciferase reporters, at the same time Torin (250 nM) or DMSO were added; 12 h after Dox addition, lysates were harvested in passive lysis buffer (Promega). Luciferase activity for both FLuc and RLuc was analyzed using the Dual-Luciferase Reporter Assay (Promega) and normalized (FLuc/RLuc) relative luciferase was calculated in relative light units (RLUs). Results were plotted using GraphPad, an unpaired t-test was performed, ±SEM; n = 6, *** = P<0.001. D&E: HeLa Tet-Off cells were transduced with recombinant lentiviruses expressing either shRNAs targeting Atg5 or Atg14 (shAtg5, shAtg14) or a non-targeting control (NS) and selected with puromycin (1 μg/mL). After selection, cells were co-transfected and Dox treatment was performed as described in C, except that co-transfection also included an expression plasmid for KapB or an empty vector control. Results were plotted using GraphPad, an unpaired t-test was performed, ±SEM; n = 3, * = P<0.05, ** = P<0.01. F: Cells were co-transfected as in E and BafA1 (10 nM) was added at the same time as Dox. Results were plotted in GraphPad and a Student’s t-test was performed, ±SEM; n = 3, * = P<0.05, ** = P<0.01.

Article Snippet: HEK293T cells (ATCC), HeLa Tet-Off cells (Clontech), Atg5 +/+ and -/- MEFs [ ], HeLa Flp-In TREx GFP-Dcp1a cells (a generous gift from Anne-Claude Gingras), and iSLK.219 cells (a generous gift from Don Ganem) [ ] were cultured in DMEM (Thermo Fisher) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Thermo Fisher) and 10% FBS (Thermo Fisher). iSLK.219 cells were additionally cultured in the presence of puromycin (10 μg/mL).

Techniques: Transduction, Recombinant, Expressing, Plasmid Preparation, Infection, Lysis, Transfection, Luciferase, Activity Assay, Reporter Assay, Selection, Cotransfection

Journal: PLOS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies

doi: 10.1371/journal.ppat.1011080

Figure Lengend Snippet: A: HUVECs were sequentially transduced, first with recombinant lentivirus expressing KapB or an empty vector control and selected with blasticidin (5 μg/mL), and second with recombinant lentivirus expressing shRNAs targeting NDP52 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). Coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 and immunostained for Hedls (PBs; white), DAPI (nuclei, blue). Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, * = P<0.05. B: HUVECs were transduced with recombinant lentivirus expressing an shRNA targeting NDP52 and selected with puromycin (1 μg/mL). Cells were treated with Torin (250 nM) or a DMSO control for 2 h prior to fixation in 4% paraformaldehyde and permeabilization in 0.1% Triton X-100. Samples were immunostained for Hedls (PBs; white), DAPI (nuclei, blue). Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, **** = P<0.0001. C: HUVECs were transduced with recombinant lentiviruses expressing either an shRNA targeting NDP52 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). Cells were treated with DMSO or Torin (250 nM) for 4 h prior to harvest in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a, Pat1b or DDX6. Samples were quantified by normalizing Dcp1a or Pat1b protein levels to the total protein in each lane using Image Lab (BioRad) and then to the NS DMSO control. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3 (Dcp1a) n = 4 (Pat1b), * = P<0.05. D: HeLa Tet-Off cells were transduced with recombinant lentivirus expressing shRNAs targeting NDP52, OPTN, p62 or a NS control and selected with puromycin (1 μg/mL) then cells were co-transfected, treated with Dox and luciferase activity was recorded and analyzed as in . Data were plotted in GraphPad as the mean fold change in the relative luciferase activity of each condition compared to vector NS or KapB NS; n = 4. An unpaired t-test was performed; * = P<0.05 ** = P<0.01. E: HUVECs were sequentially transduced first with recombinant lentivirus expressing shNDP52 targeting the 3’-UTR of NDP52 or a NS control and selected with blasticidin (5 μg/mL), and second, with recombinant lentivirus expressing KapB and either mCherry control (mCh) or RFP-NDP52. Coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and immunostained with Hedls (PBs, green). Scale bar = 20 μm. F: Samples from E were quantified; Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, **** = P<0.0001.

Article Snippet: HEK293T cells (ATCC), HeLa Tet-Off cells (Clontech), Atg5 +/+ and -/- MEFs [ ], HeLa Flp-In TREx GFP-Dcp1a cells (a generous gift from Anne-Claude Gingras), and iSLK.219 cells (a generous gift from Don Ganem) [ ] were cultured in DMEM (Thermo Fisher) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Thermo Fisher) and 10% FBS (Thermo Fisher). iSLK.219 cells were additionally cultured in the presence of puromycin (10 μg/mL).

Techniques: Recombinant, Expressing, Plasmid Preparation, Transduction, shRNA, SDS Page, Western Blot, Transfection, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: DNA–protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM)

doi: 10.1093/nar/gkac1214

Figure Lengend Snippet: Formation of TFAM-DPCs in mitochondria of HeLa cells. ( A ) Confirmation of the expression of Myc-tagged UNG1-Y147A in mitochondria of HeLa cells by western blotting. Tom20 was used as a loading control. UNG1-Y147A contained a Myc tag. Treatment conditions were indicated by two-letter codes. The first letter indicates whether BSO is present, with N denoting untreated cells and B denoting BSO-treated cells. The second letter indicates whether the cells were treated with doxycycline, with D denoting doxycycline-induced expression of UNG1-Y147A and N denoting untreated cells. ( B ) Quantification of AP sites in mtDNA using ARP assays. ( C ) mtDNA copy-number by RT-PCR, normalized by the nDNA copy number based on the β-globin gene. ( D ) Quantification of total cellular glutathione. ( E ) The fold-change of TFAM-DPCs relative to untreated cells based on ELISA. ( F ) Purity of mtDNA in the assays. NT, nontreated mtDNA from isolated by differential centrifugation; NC, negative control, mock reactions without turbonuclease; turbo, turbonuclease treatment. Data were from independent experiments as indicated and were mean ± S.D. (or range of data for NN and NC in panel F). For NN in (E), the fold-change was normalized to control in each independent experiment, and therefore NN represents the mean from five independent experiments without showing the error bar. * indicates P < 0.05; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates no significant difference.

Article Snippet: Tet-on HeLa cells (Takara, 631183) were transduced with a lentiviral vector encoding tetracycline-inducible mitochondrially targeted human UNG1-Y147A (Addgene plasmid 46883).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, Negative Control

Journal: bioRxiv

Article Title: Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

doi: 10.1101/2022.12.29.522256

Figure Lengend Snippet: Effects of EN inhibitors on L1-induced DNA damage. HeLa Tet-On cell lines containing doxycycline-inducible FL or EN domain only L1 expression constructs were generated for WT and mutant (EN-, H230A; RT-, D702Y) L1. A) Representative immunofluorescence images of HeLa cells with indicated constructs stained for γ-H2AX. B) Average of mean γ-H2AX intensities of individual nuclei normalized to WT no inhibitor control for FL expression (left, n=254-752) or EN domain (right, n=56-325). Results are from at least 3 independent experiments per sample. All samples from the same experiment were processed in parallel and images were acquired with the same exposure. Mean γ-H2AX intensity analysis was performed with CellProfiler. Statistical significance of mean (red bars) vs. WT was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism version 9.4.1 for Windows. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. C) Representative images of neutral COMET assay results for HeLa cells expressing EN domain constructs and treated with inhibitors or hydrogen peroxide as indicated.

Article Snippet: HeLa Tet-On cells (Takara Bio Inc.) were cultured at 37 °C at 5% CO2 in air atmosphere.

Techniques: Expressing, Construct, Generated, Mutagenesis, Immunofluorescence, Staining, Neutral Comet Assay