Journal: bioRxiv

Article Title: Rad51 inhibition sensitizes non-replicating quiescent cells to UVB radiation and transcription stress

doi: 10.1101/2022.09.20.508657

Figure Lengend Snippet: (A) Schematic of DSB repair pathways activated in response to NER-dependent DSB formation. Small molecule inhibitors targeting the indicated DNA repair protein are indicated. (B) Relative cell survival as measured by MTT assay 3 days after treatment of quiescent HaCaT cells with the indicated inhibitor and exposure to 400 J/m 2 UVB radiation. One-way ANOVAs were used to compare the relative survival of the different treatment groups from at least two independent experiments (*, p<0.05; **, p<0.01; ***, p<0.001). (C) Quiescent HaCaT cells were treated with vehicle (0.1% DMSO) or 10 μM Rad51 inhibitor (Rad51i B02) for 30 min before exposure to the indicated fluence of UVB radiation. Cell survival was measured by trypan blue staining 24 hr later, and the percentage of trypan blue-negative cells are indicated. (D) Cells were treated as in (C) except that whole cell lysates were prepared 12 hr after UVB exposure for immunoblot analysis of apoptotic signaling. (E) Clonogenic survival of quiescent HaCaT cells treated with vehicle or Rad51i and exposed to UVB. Cells were re-plated in normal medium 4 days later and then stained and counted 10-14 days later. (F) Immunoblot analysis of Rad51 protein levels in proliferating and quiescent HaCaT cells. (G) Immunoblot analysis following transfection with Control (siControl) siRNA or a pool of siRNAs targeting Rad51 (siRad51). (H) Relative cell survival of quiescent cells following knockdown of Rad51 and exposure to UVB. (I) Relative survival was determined by MTT assay in quiescent HaCaT cells treated with DMSO or Rad51i and then exposed to 400 nM Illudin S or 60 μM cisplatin for 3 days. (J) Quiescent HaCaT cells treated with DMSO or the Rad51i were exposed to 200 J/m 2 UVB radiation and then fractionated to enrich for chromatin-associated proteins, which was examine by immunoblotting. (K) Immunofluorescence microscopy was used to examine Rad51 nuclear staining in quiescent HaCaT cells in the absence and presence of UVB exposure and after treatment with the Rad51i B02.

Article Snippet: Rad51 was knocked down using a pool of Rad51 siRNAs (Santa Cruz sc-36361) and siRNA transfection reagent (Santa Cruz Biotechnology).

Techniques: MTT Assay, Staining, Western Blot, Transfection, Control, Knockdown, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Rad51 inhibition sensitizes non-replicating quiescent cells to UVB radiation and transcription stress

doi: 10.1101/2022.09.20.508657

Figure Lengend Snippet: (A) Schematic of DSB repair pathways activated in response to NER-dependent DSB formation. Small molecule inhibitors targeting the indicated DNA repair protein are indicated. (B) Relative cell survival as measured by MTT assay 3 days after treatment of quiescent HaCaT cells with the indicated inhibitor and exposure to 400 J/m 2 UVB radiation. One-way ANOVAs were used to compare the relative survival of the different treatment groups from at least two independent experiments (*, p<0.05; **, p<0.01; ***, p<0.001). (C) Quiescent HaCaT cells were treated with vehicle (0.1% DMSO) or 10 μM Rad51 inhibitor (Rad51i B02) for 30 min before exposure to the indicated fluence of UVB radiation. Cell survival was measured by trypan blue staining 24 hr later, and the percentage of trypan blue-negative cells are indicated. (D) Cells were treated as in (C) except that whole cell lysates were prepared 12 hr after UVB exposure for immunoblot analysis of apoptotic signaling. (E) Clonogenic survival of quiescent HaCaT cells treated with vehicle or Rad51i and exposed to UVB. Cells were re-plated in normal medium 4 days later and then stained and counted 10-14 days later. (F) Immunoblot analysis of Rad51 protein levels in proliferating and quiescent HaCaT cells. (G) Immunoblot analysis following transfection with Control (siControl) siRNA or a pool of siRNAs targeting Rad51 (siRad51). (H) Relative cell survival of quiescent cells following knockdown of Rad51 and exposure to UVB. (I) Relative survival was determined by MTT assay in quiescent HaCaT cells treated with DMSO or Rad51i and then exposed to 400 nM Illudin S or 60 μM cisplatin for 3 days. (J) Quiescent HaCaT cells treated with DMSO or the Rad51i were exposed to 200 J/m 2 UVB radiation and then fractionated to enrich for chromatin-associated proteins, which was examine by immunoblotting. (K) Immunofluorescence microscopy was used to examine Rad51 nuclear staining in quiescent HaCaT cells in the absence and presence of UVB exposure and after treatment with the Rad51i B02.

Article Snippet: Rad51 was knocked down using a pool of Rad51 siRNAs (Santa Cruz sc-36361) and siRNA transfection reagent (Santa Cruz Biotechnology).

Techniques: MTT Assay, Staining, Western Blot, Transfection, Control, Knockdown, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Rad51 inhibition sensitizes non-replicating quiescent cells to UVB radiation and transcription stress

doi: 10.1101/2022.09.20.508657

Figure Lengend Snippet: (A) Representative image showing Comet tails in WT and XPA-KO cells exposed to the indicated fluence of UVB radiation. (B) Quantitative analysis of Comet tail moment from 50 individual cells analyzed as in (A) with by OpenComet software. An ordinary one-way ANOVA was used to compare treatment groups (***, p<0.001; n.s., not significant). (C) Immunofluorescence microscopy was used to examine Rad51 nuclear staining in quiescent XPA-KO HaCaT cells in the absence and presence of UVB exposure. (D) XPA-KO cells were treated as indicated and then fractionated to enrich for chromatin- associated proteinss. (E) MTT assays were performed 3 days after exposure of quiescent XPA-KO cells treated with DMSO or Rad51i to the indicated fluences of UVB radiation. (F) Quiescent HaCaT cells were treated with vehicle or the NER inhibitor spironolactone (SP) in the absence or presence of Rad51i before exposure to the indicated fluences of UVB radiation and measurement of cell survival by MTT assay.

Article Snippet: Rad51 was knocked down using a pool of Rad51 siRNAs (Santa Cruz sc-36361) and siRNA transfection reagent (Santa Cruz Biotechnology).

Techniques: Software, Immunofluorescence, Microscopy, Staining, MTT Assay

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) Flow-cytometry quantification of telomere vesicles released by 10 7 APCs in culture media following the indicated treatments, with or without ionomycin for 18h. ( b ) Shelterin down-regulation in APCs following activation with ionomycin as in ( a ). Representative POT1 and TRF2 staining’s ( left ) and pooled data (n = 2; rights ) are shown. Each dot is an individual cell. Scale bar, 2 μm. ( c ) APCs donate shelterin-devoid telomeres. APCs were live-labelled with TelC telomere probes, activated with ionomycin, then analysed by IF to shelterin (POT1). Arrows indicate ‘shelterin-devoid’ telomeres released by APCs. Scale bar, 10 μm. Representative microscopy field ( left ) and pooled data from 30 fields ( right ) are shown. ( d ) APCs were transduced with mock or shelterin expressing (TRF2 + POT1) vectors, and labelled with BrdU. APCs were then activated 18h by ionomycin and telomere vesicles released by APCs were immunoprecipitated with anti-BrdU from cell-free supernatants. Representative blots ( bottom ) and data form three experiments ( top ) are shown. See also Extended Data Fig.9. ( e ) Telomere vesicles were analysed by IF to Rad51. Individual staining’s ( left ) and quantification from 42 microscopy fields is shown ( right ). Scale bars, 2 μm. ( f ) APCs were transfected with siCtrl or siRad51 for 36h, live-labelled with Cy3-PNA telomere probes then activated by ionomycin for 18h. The cell-free supernatants containing fluorescent APC-telomere vesicles were then transferred into T cells with FITC-PNA telomere labelling. There was no contact between APCs and T cells. APC-T cell telomere co-localization (Cy3 + FITC = yellow) in recipient T cell nuclei was quantified by confocal imaging 24h later. Representative images ( left ) and pooled data from 6 experiments ( right ) are shown. Arrowheads indicate APC-T cell telomere co-localization. Scale bar, 5 μm. In ( a , b, c, d, f ), Mann-Whitney test. *p<0.05 *** P < 0.001. Error bars indicate S.E.M. throughout.

Article Snippet: APCs were nucleofected with siCtrl (10 nM; sc-37007, Santa Cruz) or siRad51 (10 nM; sc-36361, Santa Cruz), labelled with Cy3-TelC PNA probes and extracted as above described.

Techniques: Flow Cytometry, Activation Assay, Microscopy, Transduction, Expressing, Immunoprecipitation, Transfection, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) Immunoblot analysis of the indicated DNA damage factors in EV pellets derived by sequential centrifugation of APC supernatants following activation with ionomycin for 18h. APCs served as whole cell lysate control. Representative of 3 separate experiments. ( b ) Immunoblots of Rad51 following indicated siRNA treatment in human APCs. H2B served as loading control. The numbers indicate knockdown efficiency. Representative of 3 separate experiments. ( c ) FESEM analysis of 200 siRad51 telomere vesicles purified by fluorescence activated vesicle sorting among cell-free supernatants of APCs transfected as in ( b ) then activated with ionomycin for 18h. ( d ) Protein cargo in siRad51 or siCtrl telomere vesicles by indirect ELISA. Telomere vesicles (250 per sample, in triplicate wells) were purified by fluorescence activated vesicle sorting from cell-free supernatants of APCs transfected with either siCtrl or siRad51 short-interference RNA, then stimulated with ionomycin for 18h. Pooled data from 4 experiments are shown. ( e ) Metaphase Q-FISH coupled to TFL software analysis showing elongation of individual T cell chromosome ends upon transfer of telomere vesicles derived from APCs. Each dot represents an individual T cell chromosome with APC-derived telomeres; 370 (siCtrl) and 378 (siRad51). ( f ) TRF analysis comparison of siCtrl vs siRad51 EVs isolated by 100,000g ultracentrifugation following activation with ionomycin for 18h. Pooled data from 3 experiments are shown ( right ). ( g ) Reduced fusion (elongation) between siRad51 telomere vesicles and T cell telomeres. Recipient T cells were treated with 5,000 FACS-purified telomere vesicles (Tel + ) from siCtrl or siRad51 APCs and total nuclear T cell extracts were analysed by qPCR 48h later. 5,000 telomere depleted vesicles (Tel - ) were also transferred as control. ( h ) Pooled data (n=3) showing reduced single-strand telomeric DNA in siRad51 vs siCtrl telomere vesicles by quantitative amplification of single-stranded DNA (QAOS). In ( d, h ) student’s t test, in Mann-Whitney test, in ( g ) Anova with Bonferroni post-correction test. *p<0.05, **p<0.001 *** P < 0.001. Error bars indicate S.E.M. throughout.

Article Snippet: APCs were nucleofected with siCtrl (10 nM; sc-37007, Santa Cruz) or siRad51 (10 nM; sc-36361, Santa Cruz), labelled with Cy3-TelC PNA probes and extracted as above described.

Techniques: Western Blot, Derivative Assay, Centrifugation, Activation Assay, Control, Knockdown, Purification, Fluorescence, Transfection, Indirect ELISA, Software, Comparison, Isolation, Amplification, MANN-WHITNEY

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) T cells were activated with anti-CD3 plus anti-CD28 every 10 days in the absence or presence of 1,000 siCtrl or siRad51 telomere vesicles (Tel + ) purified from APCs by FACS. Control T cells were cultured with 1,000 telomere depleted vesicles (Tel - ) or left without any vesicles (no vesicles) throughout. Population doublings, PD. The experiments were stopped after 39 days at which point the control T cells stopped dividing (∼10 PD). ( b ) Expansion of human T cells by heterologous telomere vesicles. Control T cells were cultured 10 days in the presence of 1,000 telomere vesicles (Tel + ) or telomere depleted vesicles (Tel - ) purified by FACS as in ( a ) and derived from either mismatched human (h) or mouse (h) APCs, as indicated. T cells cultured without any vesicles are also shown. Each dot is an independent T cell culture. ( c ) Senescence-associated beta-galactosidase activity was measured in T cells activated as in ( a ) for 10 days. Four donors. ( d ) OVA-pulsed APCs with Cy-3 TelC labelled-telomeres were injected into the right footpad of recipients followed by adoptive-transfers of OT-II cells (identified by Cell Trace Violet dye). Animals were culled 18h after the addition of T cells and popliteal lymph nodes collected. The frequencies of donor OT-II cells with APC-derived telomeres were calculated by flow-cytometry. In control experiments, APCs with fluorescently-labelled telomeres were not loaded with OVA antigens. Representative plot ( left ) and results from 3 experiments are shown ( right ). ( e ) Presence of APC-derived telomeres into the nuclei of mouse T cells (metaphase spreads) following in vivo adoptive transfer experiments performed as in ( d ). Scale bar, 5 μm. ( f ) GFP + (donor) OT-II cells were pre-treated with or without 250 telomere vesicles derived from mouse APCs activated with ionomycin, injected into recipients, and splenocytes were analysed by flow cytometry with anti-CD4 and anti-CD62L 8 weeks post-transfer (14 days after re-challenge with OVA antigen, left ). Controls were injected with either 250 telomere depleted vesicles (Tel-) or saline solution (no vesicles). Pooled data from 3 mice per group are shown ( right ). In ( a, b, c, f ), ANOVA with Bonferroni post-test correction; in ( d ), paired student T test. *** P < Error bars indicate S.E.M. throughout.

Article Snippet: APCs were nucleofected with siCtrl (10 nM; sc-37007, Santa Cruz) or siRad51 (10 nM; sc-36361, Santa Cruz), labelled with Cy3-TelC PNA probes and extracted as above described.

Techniques: Purification, Control, Cell Culture, Derivative Assay, Activity Assay, Injection, Flow Cytometry, In Vivo, Adoptive Transfer Assay, Saline

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) Telomere vesicles were extracted with ionomycin from APCs that had been previously transfected with either siCtrl or siRad51 RNAs then transferred to T cells. T cells were then activated by anti-CD3 plus anti-CD28 for 10 days. IF demonstrated that activated T cells do not up-regulate senescence-associated sestrin proteins (sestrin 1) upon transfer of Tel + vesicles. By contrast, T cells up-regulated sestrins (albeit to a lesser extent) upon transfer of siRad51 Tel + vesicles. Two donors. In ( b ), Kruskal-Wallis with Dunn’s post-correction test. *P < 0.05; ***P < 0.001 .

Article Snippet: APCs were nucleofected with siCtrl (10 nM; sc-37007, Santa Cruz) or siRad51 (10 nM; sc-36361, Santa Cruz), labelled with Cy3-TelC PNA probes and extracted as above described.

Techniques: Transfection