EMBO/SourceData huggingface dataset:
Figure 2A. Concentration of the various indicated isotypes in the serum of 6-8 week old unimmunized WT and Shld2−/− mice. Values are mean concentration ± SD of 4 biological replicates; ** P ≤ 0.01, unpaired two-tailed t-test.B. B cells were purified from spleens from WT and Shld2−/− mice, and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails [33]. Cells were then analyzed by flow cytometry for expression of the various indicated isotypes and the percent expression of each isotype are reported. Values are mean frequency ± SD of 4 biological replicates; **** P ≤ 0.0001, unpaired two-tailed t-test.C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates.D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated and anti-IgG secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2-/- (n=3); * P ≤ 0.05, ** P ≤ 0.01, unpaired two-tailed t test.
Figure 3A. WT and Shld2−/− B cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3, 6, and 9 days post-stimulation by flow cytometry. Representative plots are shown for both WT and Shld2−/− B cells 6-days post stimulation. The graph plots show proportion of IgG1+ and Iglo cells, mean ± SD from 6 biological replicates; ** P ≤ 0.01, *** P ≤ 0.001, two-way ANOVA with post hoc Dunnett's test.B. WT CH12 cells, as well as two each of 53BP1-, SHLD1-, SHLD2-, and SHLD3-deficient clones generated previously [21], as well as a LIG4-deficient CH12 clone were subjected to CSR induction with the CIT cocktail and measured for both IgM and IgA expression by flow cytometry. Representative flow plots for WT and 53bp1−/− CH12 cells are shown at day 3 post CIT stimulation. The graph plots show proportion of IgA+ and Iglo CH12 cells, mean ± SD from 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. The letters below the x-axis represent the clone codes for the 53BP1, SHLD1, SHLD2, and SHLD3-deficient CH12 clones. One LIG4-deficient CH12 clone was also used.C. The Xlf, Ku70, Ku80, Xrcc4, and Paxx genes were knocked out in CH12 cells by CRISPR, and two independent clones each were analyzed as in Figure 3B with mean ± 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.
Figure 4A. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression. The Iglo population were reduced after 7 days in culture. Values are mean ± SD from 3 biological replicates; * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.B. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort (see Figure EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post CIT treatment is shown.
Figure 5A. mRNA expression analysis of IgM and IgA was carried out by RT-PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT-PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (bottom) as control.B. mRNA expression analysis as in A of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones, and IgA+ subclones from Shld2-/-/- clone F3. The Iglo subclones derived from the Shld2-/-/- CH12 F2 clone are listed as H03-H40, while the Iglo subclones derived from the Shld2-/-/-CH12 F3 clone are listed as J02-J109.
Figure 6Long-range PCR of Iglo subclones from WT and two independent 53bp1-/- CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show long-range amplicons (top) and AID genomic DNA PCR control (bottom).Long-range PCR as in B of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones (left 2 panels), and IgA+ subclones from Shld2-/-/- clone F3.Sequence analysis of a representative subset of long range-PCR amplicons. Symbol annotations: (*) inverted sequence, (◊) rearranged fragment order, (†) amplified with the EμF primer rather than IμF, (‡) undamaged Cα coding sequence.
Figure 1A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated.B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis.C. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C) or vehicle.D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of AMG517 (D) or vehicle.E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1-/-mice.F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of saline or vehicle.
Figure 2A. Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1-/-mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left.B. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice, and a bar graph highlighting the response at 24 h.C. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1-/-mice, and bar graph highlighting the response at 24 h.D. Hematoxylin and eosin stained tissue sections of iodine treated ear in 0.15% Oxa-challenged WT and TRPA1-/-mice (n ≥ 3).E. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1-/-mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h.F. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h.
Figure 3A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit.B. Concentration-response relationships of iodine-induced intracellular Ca2+ increase in HEK293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation.C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031D. Concentration-response relationship of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. Data are presented as mean ± s.e.m. n ≥ 8 for each concentration. The smooth curve is a fit to the Hill equation.E. Time course of intracellular iodine-induced macroscopic currents in an inside-out patch from hTRPA1-expressing HEK293 cell (n = 3).F. Time course of iodine-induced currents in a hTRPA1-expressing Xenopus oocyte, which presents the current reduction upon DTT treatment (n = 3).G, H. Comparisons of the iodine-induced whole-cell current in HEK293 cells expressing WT hTRPA1 (G) or the triple mutant channel (C421S/K710R/C856S) (H) (n ≥ 4).I. Representative whole-cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).
Figure 1A Growth curves show proliferation reduction in ATRX depleted U87 cells. Data are expressed as means ± standard deviation (s.d.), N = 2. 500 cells/well were plated.B β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 µm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± s.d., N = 3, unpaired t test.C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87 cells (D). Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.E C-circle assays revealed enhanced C-circles formation in ATRX-deleted U87 cells. Reactions lacking DNA or ɸ29 serve as negative controls. 30 ng of indicated template DNA was used in each reaction.53BP1/TelG immuno-FISH (F)G 53BP1/TelG immuno-FISH shows increased TIF formation in ATRX-deleted U87 cells (G). Arrows denote telomeres positive for 53BP1 signals. Scale bar, 10 µm. (G) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.H Growth curves revealed increase of cell proliferation in ATRX-deleted U87 cells (A-1 and A-2) after hTERT transduction. Data are expressed as means ± s.d., N = 2.I Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-1 and A-2 U87 cells.
Figure 2A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT-overexpressed U87-T cells, but further removal of the exogenous floxed TERT by Ad-Cre induced rapid cell cycle arrest. Data are expressed as means ± s.d., N = 3.B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control.C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87-T cells (D) at day 9 post control or Ad-Cre infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.E 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA.F C-circle assays shows increased C-circle formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection.G, H 53BP1/TelG immuno-FISH (G) shows increased TIF formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (H). Scale bar, 10 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.
Figure 3B Western blots of extracts from U87 cells infected with Lenti-CRISPR(puro) encoding individual ATRX-targeted sgRNAs. The extracts were isolated after puromycin selection, and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control. The asterisks (*) denote the two sgATRXs (sgA#10 and sgA#19) used in following experiments.C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa, sgA#10, or sgA#19. D, DAPI. Scale bar, 50 µm.Bar graphs show time-course analyses of U87 (E) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.Bar graphs show time-course analyses of U2OS cells (F) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.G, H 53BP1/TRF2 co-immunofluorescence (G) revealed strongly increased TIF formation in lentiviral sgA#10 or sgA#19 transduced U87 cells assayed at day 45 but not day 15 post infections (H). Scale bar, 15 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.I, J 53BP1/TelG immuno-FISH (I) revealed strongly enhanced TIF formation in lentiviral sgA#10 or sgA#19 transduced IMR90 cells assayed at day 35 but not day 10 post infections (J). Scale bar, 10 µm. (J) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.K Bar graphs show time-course analyses of IMR90 cells transduced with lentiviral sgRosa, sgA#10 or sgA#19. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.
Figure 4A, B γH2AX/TelG immuno-FISH (A) revealed increased TIF formation in IMR90 cells transduced with DAXX-targeted lentiviral sgRNAs (sgD#1 and #2) (B). Scale bar, 10 µm. Arrows denote telomeres that are positive for γH2AX signals. (B) Percentage of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.C Western blots show depleted DAXX protein expression in Ad-Flp-infected cd-DAXX cells.D, E PML/TelG immune-FISH (D) show enhanced APB formation in Ad-Flp infected cd-DAXX cells (cd-D#1 and cd-D#2) (E). The cells were analyzed at day 18 post Ad-Flp infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (E) Percentages of APB+ cells are expressed as means ± s.d., N = 3, unpaired t test.Depletion of DAXX induced delayed onset of growth arrest (F) (F) 2,000 cells were plated at day 4 post Ad-Flp infection. Data are expressed as means ± s.d., N = 3.Depletion of DAXX induced C-circle formation (G).
Figure 5IMR90 cells at day 25 post infection of lentiviral control or sgA#10 were synchronized by serum starvation and switched to 15% serum-containing medium and assayed by 53BP1/TelG immuno-FISH (A) at indicated time. Arrows denote telomeres that are positive for 53BP1 signals. Scale bar, 15 µm. (B) Percentage of cells containing ; 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.C, D RPA/TRF2 co-immunofluorescence (C) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA#10 infection (D). Lentiviral control and sgA#10 infected U87 cells at indicated time post-infection were released following thymidine (Thy) synchronization and processed for RPA and TRF2 co-staining. Scale bar, 10 µm. (D) Percentages of cells with > 4 RPA/TRF2 co-localized foci are expressed as means ± s.d., N = 3, unpaired t test.
Figure 6A, B ATAC-seq analyses revealed progressive increased telomeric DNA accessibilities in sgA#10 transduced IMR90 cells. (A) The graph plots show the cumulative percentage of total reads that contained at least indicated number of TTAGGG (or CCCTAA) tandem repeats. (B) The percentage of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s.d., N = 2, unpaired t test.C, D ChIP assays (C) show reduced nucleosome densities in sgA#10 transduced IMR90 cells (D). Input and ChIP DNAs processed against indicated antibodies were assayed by dot-blotting and hybridized with 32P-labeled probes specific for TelG. (D) The relative enrichment was calculated after normalization of ChIP DNA signals to the respective input DNA signals. Data are expressed as means ± s.d., N = 3, paired t test.
Figure 7A Growth curves show the immortalization processes of lentiviral Rosa control (sgRosa#1 or #2) or sgATRX (sgA#10 or #19) transduced IMR90 cells. TERT transduced IMR90 culture was included as a positive control for immortalization.B Quantitative RT-PCR of endogenous TERT gene expression revealed that sgATRX but not sgRosa transduced IMR90 cells adopted ALT during immortalization. Data are expressed as means ± s.d., N = 3, unpaired t test.C Western blots show depletion of ATRX protein expression in the immortalized ALT IMR90 cells.D C-circle analysis shows increased C-circle formation in the immortalized ALT cells. U2OS was used as a positive control, and reactions lacking DNA or ɸ29 serve as negative controls.E Telomere restriction fragment (TRF) analysis show enhanced telomeric length heterogeneities in the immortalized ALT cells.F, G PML/TRF2 co-immunofluorescence (F) shows increased APB formation in the immortalized ALT cells (G). Scale bar, 10 µm. (G) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.
Figure 1(A) Kaplan Meier plot for survival in NeuT;Apln+/+ (n=11) and NeuT;Apln-/- (n=10) mice with mammary cancer after tumor onset. *P=0.0185; Log rank test.(B) Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically-injected into both syngeneic C57BL/6J Apln+/+ and Apln-/- mice (5x105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S.E.M. Data shown is pooled from 2 independent experiments. Apln+/+;shRenilla (n=18), Apln+/+;shApln (n=17), Apln-/-;shRenilla (n=14), Apln-/-;shApln (n=15); ** P<0.01, *** P<0.001; two-way ANOVA.(C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm(E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels).(F) Mean percentage (± S.E.M.) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n=8) or shApln (n=6) were orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice (5x105 cells/mouse), respectively and tumors were harvested day 25 post-injection. *P<0.05, **P<0.01; t test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells, (Thy1.2+, CD4+), Inflammatory Monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+) and peripheral dendritic cells (Ly6G- ,Ly6C+, Cd11b- , PDAC1+, B220+).
Figure 2(A) Left - Heatmap of RNA-Seq transcriptome analysis of CD31+/CD105+ endothelial cells sorted from tumors established by E0771 shRenilla (n=6) or shApln (n=3) cells orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice, respectively. Tumors were harvested day 25 post-injection and genes displayed are significantly deregulated at the adjusted p-value cut-off of 0.05. Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes.(B) Quantification of vessel sprouts (mean values ± S.E.M.) upon VEGF treatment (30ng/mL) of embryoid bodies (EB) derived from murine ES cells (mESCs) with sense integrations in the Apelin gene (Apln STOP) of the splice acceptor described in Appendix Figure S1B or Cre-reverted antisense (Apln GO) sister cells. Apln GO (Day 2 n=6; Day 4 n=40, Day 6 n=54, Day 8 n= 32, Day 10 n=35, Day 11 n=31), Apln STOP (Day 2 n=4; Day 4 n=15, Day 6 n=31, Day 8 n= 28, Day 10 n=32, Day 11 n=37); ** P<0.01, *** P<0.001, two-way ANOVA. Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels. Scale bars = 200 μm(C) Differentially expressed genes using RNAseq transcriptome analysis of CD31+ endothelial cells (ECs) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF (30ng/mL) and DMSO (-Apln) and repaired Apelin GO sister cells stimulated with VEGF and Apelin (1000nM) (+Apln). VEGF target genes and angiogenesis-related genes, predicted by Ingenuity Pathway Analysis (IPA) software, are indicated by bars on the upper axis of the heatmap. GO terms were analyzed by DAVID on-line software.