EMBO/SourceData huggingface dataset:
Figure 2(A) Telomere length analysis of cells from different sequential restreaks following introduction of Poz1-V5-Rap1_491-693 into rap1∆ cells. Telomere length was assessed by Southern blotting of EcoRI-digested genomic DNA probed with a telomere specific probe. (B) PFGE analysis of nitrogen-starved Poz1-V5-Rap1_491-693 cells after 5 and 14 restreaks.(C) Telomere length analysis of cells with Rap1∆RCT fused to Taz1 or Poz1 with intervening V5 tag. The fusion protein was integrated at the endogenous rap1 locus in the context of taz1∆ or poz1∆, respectively. Different sequential restreaks following introduction of the fusion construct were analyzed. (D) PFGE analysis for nitrogen-starved Rap1∆RCT-V5-Taz1 and Rap1∆RCT-V5-Poz1 from 2nd restreak.
Figure 4(B) PFGE analysis of cells containing plasmids of Rap1_440-693 with mutations of the amino acids identified in (A) in the context of rap1∆. Cells were subjected to nitrogen-starvation after recovery from transformation (Restreak 0). (C) PFGE analysis of nitrogen-starved cells harboring the same mutations after 5 restreaks on plates (approximately 110 generations). (D) Quantification of (B) and (C) using Fiji. The y-axis shows the percentage of intensity represented by the three fusion bands (I+L, I+M and L+M) relative to the total intensity of the fused and unfused signals. WT: wildtype Rap1_440-693.
Figure 5(B) Western blot analysis of N-terminally V5-tagged truncation mutants using anti-V5 antibody. Anti-α-tubulin was used as loading control. Quantification of Rap1 mutant protein levels relative to WT normalized to α-tubulin is shown beneath the gel.(C) Telomere length analysis of the mutants by Southern blot with telomere specific probe. All mutants are V5-tagged except for ∆491-638 untagged (lane 7).(D) PFGE analysis of the same strains in (C) that were nitrogen-starved. (E) PFGE analysis of nitrogen-starved internal deletion mutants in poz1∆ background.
Figure 6(A) PFGE analysis of nitrogen-starved N-terminally V5-tagged full length Rap1 mutants. All mutants were integrated at the endogenous rap1 locus. M5A is the mutation of all 5 non-alanine amino acids under selection in the p-patch to alanine. (B) PFGE analysis of nitrogen-starved N-terminally V5-tagged rap1_440-693 mutants integrated at the endogenous rap1 locus.(C) PFGE analysis of nitrogen-starved rap1 mutants in poz1∆ background.(D) PFGE analysis of nitrogen-starved rap1 mutants in ppm1∆ background. Untagged Rap1 are in lanes 1-2 and N-terminally V5-tagged in lanes 3-7.
Figure 7(A) ChIP-qPCR analysis of N-terminally V5-tagged WT, F545L and M6 mutants of Rap1. Untagged Rap1 was used as a control. Bars represent mean of percentage of DNA precipitated with anti-V5 antibody conjugated beads relative to input DNA. Error bars represent SEM. N= 4 biological replicates. Two-tailed unpaired Student's t-test was performed and significance is indicated. ***: p < 0.001, **: 0.001 ≤ p < 0.01, n.s.: p>0.05. (B) ChIP-qPCR analysis of N-terminally V5-tagged WT, F545L and M6 mutants of Rap1 in poz1∆ background performed as in (A). N= 4 biological replicates. Statistical analysis is the same as (A).(C) Single-stranded overhang measured by denaturing gel electrophoresis of duplex-specific nuclease digested genomic DNA and Southern blot with oligonucleotide probes specific for the G-strand (top panel) and C-strand (bottom panel). WT, F545L and M6 mutants of Rap1 in the presence or absence of poz1 or ppm1 were analyzed. A 21-mer of (GGTTACA)3 was used as a positive control and size marker for G-strand and a (TGTAACC)3 oligo was used for the C-strand.
Figure 8(A) Schematic of scoring matrix. Each nucleus was divided into three concentric zones of equal area with zone I corresponding to the nuclear periphery. The focal plane with the brightest GFP signal and well-defined NE was chosen for measurements. Distance of the GFP spot to NE (X) and diameter of the nucleus were measured for the calculation. GFP signals were allocated to one of the three concentric zones of equal surface area as described (Hediger et al., 2002). Zone I: X <0.184 x radius (r)); Zone II: X between 0.184 and 0.422 times r; and X >0.422 x r is zone III. Representative images of cells expressing Nup44-mCherry (magenta) and Taz1-GFP or Pot1-GFP (green), respectively. The z-plane showing the brightest GFP spot is shown and was used for quantification. Scale bar: 2μm.(B) Stacking bar chart showing telomere distribution (labeled by Pot1-GFP) in each of the three zones. 100 telomere foci were quantified per sample. Chi square tests were performed by comparing the rap1 mutants to WT or bqt4∆. Significance is indicated. ***: p < 0.001.(C) Yeast two hybrid assay probing interaction between Rap1 and Bqt4. WT and mutants of Rap1 were fused to the DNA-binding domain (BD) in pGBKT7. Bqt4 and Taz1 were fused to the Gal4 activation domain (AD) in pGADT7. Cells (5μl) carrying both plasmids at 5 x106 cells/ml were spotted onto -Leu-Trp dropout plates and -Leu-Trp-Ade-His dropout plates to select for ADE2 and HIS3 markers. "-": Empty BD or AD plasmid.(D) PFGE analysis of nitrogen-starved bqt4∆ and bqt3∆ cells in the presence or absence of poz1.(E) End fusions in bqt4∆poz1∆ and bqt3∆poz1∆ are dependent on the presence of ligase IV. PFGE analysis of nitrogen-starved cells in the presence or absence of lig4.
Figure 1(C) Kaplan-Meier plot of PFS stratifying the patients according to high or low diversity index. N=69 (33 above the cut-off, 36 below).(D) As in (C) but plotting OS. N= 72 (34 above the cut-off, 38 below).(G) Baseline plasma FKN concentrations in responders (R, n=26), progressors (PR, n=62), hyperprogressors (HPR, n=3), age-matched healthy donors (H, n=32) and patients that were not eligible for immunotherapy (NO IT, n=28). Sample sizes represent biological replicates. Error bars are shown (standard deviations, SD), and relevant statistical comparisons by Mann-Whitney U tests are shown within the graphs.(J) Kaplan-Meier plot of PFS stratifying the patients according to the FKN cut-off value identified in the ROC curve (n=42). The associated P value is shown. (K) As in (J) but plotting OS. n= 46 (17 above the cut-off, 29 below).
Figure 2(A) Secreted FKN in cell cultures of a collection of 10 LUAD and LUSC cancer cell lines quantified by ELISA. Results are presented as means ± SD. Samples were assayed in duplicates from a pool of three independent replicates. Statistical differences among groups were analyzed by ANOVA followed by Tukey's tests.(B) Tumor growth curves of 3LL-tumor bearing mice treated with recombinant FKN (rFKN), the CX3CR1 inhibitor AZD8797, a non-relevant protein (IgG2a) or saline buffer as vehicle control (WT). Arrows indicate the time of intraperitoneal injections of rFKN (blue) and AZD8797 (black). Data are expressed as means ± SD (n=6 mice per group). Statistical comparisons by ANOVA followed by Tukey´s pairwise comparison tests are provided.(G) Kaplan-Meier survival plots. Differences between the control group (WT) and the FKN group were evaluated by a two-sided log-rank test.
Figure 3(A) FKN plasma concentration at day 15 in the indicated groups of tumor-bearing mice. Data are expressed as mean ± SD from a pool of 6 mice/group. Statistical comparisons were performed by ANOVA and Tukey´s pairwise comparison tests.(D) Percentage of the indicated peripheral immune cell populations at day 15 after injection of groups of mice (n=6 mice per group) with 3LL cells overexpressing the indicated myeloid-regulating cytokines. The relevant immune populations were quantified as percentages of total leukocytes (CD45+ cells), specifying DCs (CD11c), macrophages (F4/80), Ly6C+ monocytes and Ly6C- monocytes, granulocytes (Ly6G), B cells (CD19), NKs (NK1.1), CD4 T cells (CD3 CD4) and CD8 T cells (CD3 CD8). Relevant statistical comparisons were performed by Wilcoxon´s test followed by pair-wise comparisons of relevance by Mann-Whitney U test. Box and whisker plots indicate: median (central line), 25th to 75th percentiles (box) and minimum to maximum values (whiskers).
Figure 4(C) Kaplan-Meier survival plots of the indicated groups of mice. Survival differences between the control group (WT + αPD-1) and FKN + αPD-1 were evaluated by a two-sided log-rank test.(D) Percentage of splenic Ly6C+ monocytes, (E) Ly6G- monocytes, (F) Ly6G+ neutrophils, (G) PD-1+ T cells (CD3+), Data are presented as mean ± SD (n=6 mice per group).
Figure 5(D) Kaplan-Meier survival plot of the indicated treatment groups. Survival differences between the control group (WT) and FKN were evaluated by two-sided log-rank test.(E-J) (E) Percentage of splenic NKs (NK1.1+) expressing KLRG1, (F) TIGIT and (G) TIM3. (H) Percentage of splenic T cells (CD3+) expressing KLRG1, (I) TIGIT and (J) TIM3. Data are presented as means ± SD (n=6 mice per group). Comparisons among groups were performed by ANOVA and Tukey´s pairwise comparison tests.
Figure 1(A) Left, western blotting of PDI protein in replicative senescent hMSCs at early-passage (EP) and late-passage (LP). GAPDH was used as the loading control. Right, statistical analysis of relative PDI protein expression levels. n = 3 independent experiments. (B) Left, western blotting of PDI protein in wild-type (WT) and Werner syndrome (WS) hMSCs. GAPDH was used as the loading control. Right, statistical analysis of relative PDI protein expression levels. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(C) Left, western blotting of PDI protein in the liver of mice at young (2 months) and old (22 months) ages, GAPDH was used as the loading control. Right, statistical analysis of relative PDI protein expression levels. n = 6 mice. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(G) Left, Ki67 staining of PDI+/+ and PDI-/- hESCs. Scale bar, 25 µm. Right, statistical analysis of Ki67-positive cells. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.
Figure 2(C) Immunostaining of PDI and calreticulin (CRT) in PDI+/+ and PDI-/- hMSCs, CRT was used as ER marker. Scale bar, 25 µm.(D) Western blotting analysis of PDI family members expression in PDI+/+ and PDI-/- hMSCs at early-passage (P3) and late-passage (P8). GAPDH was used as the loading control.(G) Left, Ki67 staining of PDI+/+ and PDI-/- hMSCs at P8. Scale bar, 20 µm. Right, statistical analysis of Ki67-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(H) Left, SA-β-gal staining of PDI+/+ and PDI-/- hMSCs at P8. Scale bar, 200 µm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(K) Left, western blotting showing the P16 expression in PDI+/+ and PDI-/- hMSCs at P8. GAPDH was used as the loading control. Right, statistical analysis of relative P16 protein expression levels. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(M) Left, Immunostaining of 53BP1 and γH2AX in PDI+/+ and PDI-/- hMSCs at P8. White arrowheads indicate 53BP1 and γH2AX double positive cells. Right, statistical analysis of 53BP1 and γH2AX double positive cells. Scale bar, 10 µm. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(P) Left, Ki67 staining of PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI. Scale bar, 10 µm. Right, statistical analysis of Ki67-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(Q) Left, SA-β-gal staining of PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI. Scale bar, 200 µm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.
Figure 3(A) Oxidative folding of Myc-tagged J-Chain (JcM). PDI+/+ and PDI-/- hMSCs were transfected with JcM for 48 hr, then were pulsed with DTT and chased at indicated time after DTT removal. The lysates were analyzed using non-reducing SDS-15% PAGE and α-Myc western blotting. The mobility of reduced JcM monomers (Red), oxidized monomers (Oxi), homodimers (Dim), and high-molecular-weight (HMW) species are indicated.(D) Time-dependent changes of H2O2 levels in the ER of PDI+/+ and PDI-/- hMSCs at P8. Left, the fluorescence intensity ratio of HyPer-ER at 535 nm with excitation at 488 and 405 nm were firstly monitored at resting state for 5 min, followed by addition of 0.5 mM DTT (yellow bar) for another 15 min. Right, statistical analysis of the fluorescence intensity ratio at 488/405 nm excitation of HyPer-ER probe at 15 min post DTT addition. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(E) Analysis of the H2O2 levels in PDI+/+ and PDI-/- hMSCs at P8 using Amplex Red assays. n = 3 biological repeats. (F) Analysis of the cellular total ROS levels in PDI+/+ and PDI-/- hMSCs at P8 using H2DCFDA probe. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(M) Confocal imaging of PDI+/+ and PDI-/- hMSCs expressing Hyper7-nucleus at steady state and upon addition of 2 mM DTT. The fluorescence intensity ratio at 488/405 nm obtained from individual cells was calculated at indicated times with a representative false color image. Scale bar, 10 μm.
Figure 4(C, D) Density plot showing that upregulated (C) or downregulated (D) genes in the LP hMSCs (PDI+/+, LP vs. PDI+/+, EP) that were restored after PDI deletion (PDI-/-, LP vs. PDI+/+, LP). Comparisons were performed with two-sided Wilcoxon signed-rank test.(K) Conjoint analysis showing the secretion levels of PDI and those in (I) and various SASPs from multiple senescence inducers and in plasma proteome from 3,087 healthy individuals RS, replicative senescence (PDI+/+, LP vs. PDI+/+, EP). ATV, atazanavir treatment; RAS, inducible RAS overexpression; IR, X-irradiation. The color keys that change from dark blue to dark purple represent the fold change of gene and protein, change from white to red represent the change in protein levels per unit increase in the covariate in plasma proteomic.
Figure 5(A) qPCR analysis of SERPINE1 expression in PDI+/+ and PDI-/- hMSCs at EP and LP. n = 3 independent experiments. Data information: In (A, data are presented as mean ± SEM, two-tailed Student's t-test.(B) Western blotting analysis of SERPINE1 protein in PDI+/+ and PDI-/- hMSCs at EP and LP. GAPDH was used as the loading control of cell lysates. Ponceau was used as the loading control of conditional medium.(F) Left, Ki67 staining in PDI-/- hMSCs transduced with sgNTC or SERPINE1-targeting activation sgRNAs. Scale bar, 20 μm. Right, statistical analysis of Ki67-positive cells. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(G) Left, SA-β-gal staining in PDI-/- hMSCs transduced with sgNTC or SERPINE1-targeting activation sgRNAs. Scale bar, 200 μm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(H) qPCR analysis of the SERPINE1 mRNA expression in PDI+/+ and PDI-/- hMSCs treated with the indicated concentration H2O2 for 4 days. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(J) H2O2 levels in PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer-ER at 488/405 nm excitation after the addition of 0.5 mM DTT for 15 min. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(K) H2O2 levels in PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer7-nucleus at 500/400 nm excitation after the addition of 2 mM DTT for 20 min. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.
Figure 6(A) Western blotting of PDI expression in replicative senescent (RS) hMSCs, Hutchinson-Gilford Progeria Syndrome (HGPS) hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. GAPDH was used as the loading control.(B) Single clonal formation assay in RS hMSCs, HGPS hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.(C) SA-β-gal staining in RS hMSCs, HGPS hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. n = 3 biological repeats. Scale bar, 200 µm. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.
Figure 1A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm.B. COS-7 cells transfected with GFP-Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm.C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3).D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage.D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Western blot (WB) indicates the extent of Nup358 depletion and the level of Dcp1a.
Figure 7A. Right panel; Western blots showing the expression of transfected constructs using indicated antibodies. a-tubulin was used as loading control.A. Left panel; HeLa cells were co-transfected with GFP-MBP (control), GFP-SIM1 or GFP-SIM1-mut (AGO interaction defective) along with RL-control or RL-3xbulge. Firefly luciferase was used as transfection control. Dual luciferase assay was performed. RL/FL luminescence ration was plotted after normalization with GFP-control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.B. Bottom right panel; Western blot for monitoring the expression levels of indicated proteins using antibodies. a-tubulin was used as loading control.B. Bottom left panel; HEK293T cells were co-transfected with indicated expression constructs and the RL reporter. Firefly luciferase (FL) was used as transfection control. Dual luciferase assay was performed. The ratio of RL/FL luminescence was plotted after normalization with N-HA-MBP control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Left panel: Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage.C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Middle panel: HeLa cells were transfected with indicated siRNAs, followed by expression constructs and miRNA RL reporters. FL was used as transfection control. The ratio of RL/FL luminescence was calculated. Data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. a-tubulin is used as loading control.D. HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay. Fractions were collected and analyzed for the presence of indicated proteins by western blotting.