Fig 1: UPF1/Ku70/Ku80 recruitment to telomeres is impaired by CSL loss. a Telomere binding assay by ChIP/qPCR with antibodies against CSL and UPF1, individually or sequentially (CSL+UPF1), in parallel with non-immune IgG, in HDFs as in34,35. n(strain) = 3, *p < 0.05, two-tailed unpaired t-test. b Telomere binding assays by ChIP/qPCR with antibodies against the indicated proteins in HDFs (GB1) plus/minus siRNA-mediated CSL silencing (3 days) or CSL silencing and concomitant lentivirally induced CSL overexpression (OE). Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. 8b. c Telomere binding assays by ChIP/qPCR analysis of the same cells as in b with antibodies against TRF1/TRF2. Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. 8c. d Telomere binding assays by ChIP/qPCR with antibodies against the indicated proteins in HDFs (GB1) plus/minus lentivirally induced CSL overexpression (OE) for 3 days. Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. 8d. e Telomere binding assays by ChIP/qPCR with anti FLAG-tag antibodies in HEK293T cells expressing increasing amounts (0, 250 ng, 500 ng, and 2 μg) of FLAG-tagged full length (FL) CSL together with full length (FL) Ku70 (2 μg). Non-immune IgGs were used for normalization. A second independent experiment is in Supplementary Fig. 8e. f PLAs of stromal fibroblasts (identified by VIMENTIN staining) from unaffected skin versus flanking SCC with TRF1 or TRF2 antibodies in combination with antibodies against the other indicated proteins. Scale bar, 5 μm. Quantification of CSL and UPF1/Ku70/Ku80/TRF1/TRF2 levels in the same samples are in Fig. 2b and Supplementary Fig. 8f, respectively. Triangles, circles, and squares point to values from flanking skin (black) and corresponding SCC (red) from three patients. Non-immune IgGs were used as control. Mean ± SD, n(cells) > 77 per condition, n(SCC) = 3, n(matched Skin) = 3, *p < 0.05, two-tailed paired t-test. Bars represent mean ± SD
Fig 2: CSL binds to Ku70, Ku80, and UPF1 forming a multiprotein complex. a Co-immunoprecipitation (co-IP) analysis of HDFs with anti-CSL antibodies or non-immune IgG followed by immunoblotting with antibodies against the indicated proteins. n(strain) = 2. b Sequential co-IP analysis of HDFs with antibodies against CSL followed by IP with antibodies against UPF1 or non-immune IgG, and immunoblotting with antibodies against the indicated proteins. n(strain) = 3. c Proximity ligation assays (PLAs) of CSL and UPF1 association. HDFs with silenced CSL were used as negative control. Scale bar, 2 µm. Number of dots per cell was counted. n(cells) > 43 per condition, n(strain) = 3, *p < 0.05, two-tailed unpaired t-test. d PLAs of CSL and Ku70 and Ku80 association. Scale bar, 2 µm. Number of dots per cell was counted. n(cells) > 54 per condition, n(strain) = 3. e Binding of recombinant CSL and Ku70 proteins as measured by microscale thermophoresis (MST). Inset: thermophoretic movement of fluorescently-labeled CSL. Specificity controls are in Supplementary Fig. 5c, d. f RT-qPCR of CAF effector genes in HDFs plus/minus UPF1/Ku70/Ku80 versus CSL gene silencing for 6 days. Silencing controls are in Supplementary Fig. 6a. g RNA-seq analysis of CAF effector genes in HDFs plus/minus UPF1 versus CSL gene silencing for 7 days. Heatmap of differentially expressed genes in HDFs with CSL or UPF1 silencing relative to control is in log2 scale. Bars represent mean ± SD
Fig 3: Mapping, mutagenesis and docking analysis of CSL/UPF1/Ku70/Ku80 interaction. a Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 and 1-257, 1-464, 1-573, or 257–609 domains plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. b Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 or its 464–609 domain plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. c Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL BTD (166–334) domains plus/minus full length (FL) UPF1 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. A second independent experiment is in Supplementary Fig. 9a. d Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL point mutants (R192H, F235R, V237R, A258R, and Q307R) plus/minus full length (FL) Ku70 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. Additional information on CSL point mutants is in Supplementary Fig. 9b. e Cartoon representation showing the docking complex between CSL (cyan)—Telomere DNA (orange)—Ku70 (magenta) and Ku80 (blue) using HDOCK server. Ku70 is shown to interact with CSL-BTD domain and bind to CSL bound telomere DNA through its SAP domain, while Ku80 binds indirectly through Ku70. f Close up view of the docking complex between CSL—Telomere DNA—Ku70 showing the a-helix of C-Ku domain (olive) interacting with CSL-BTD domain (cyan), and Ku70 SAP domain (magenta) interacting with telomere DNA (orange). The “hot spot” mutations that abrogate CSL-Ku70 interaction and Ku70-telomeric DNA association as in d are shown in pink (A258R and R192H). Additional non-interfering mutations are labeled in blue (F235R, V237R, and Q307R). g–j Telomeric binding assays with antibodies against the indicated proteins followed by qPCR with telomere- and alu-specific primers in HEK293T cells expressing CSL full length (WT) and point mutants (R192H, F235R, V237R, A258R, and Q307R). Non-immune IgGs were used for normalization. Bars represent mean ± SD
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