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. 2020 Jan 14;9:e49817. doi: 10.7554/eLife.49817

Figure 6. POLD3 but not SMC5 regulates mitotic DNA synthesis at TRF1 deleted telomeres.

(A) Western blotting showing expression of SMC5, TRF1 and Actin (loading control) proteins in TRF1F/F MEFs after infection with GFP or CRE-Adenovirus and deletion of SMC5 by shRNA. shGAPDH is used as negative control. (B) Quantification of the knock-out and knock-down shown in A. Graph shows protein signal quantification relative to shGAPDH in +GFP control cells, data are represented as mean ± SEM of 3 independent biological replicates. (C) Quantification of POLD3 mRNA levels relative to GAPDH control. Data are represented as mean ± SEM of 3 independent biological replicates. (D) Representative images of 6 different genotypes generated in the above description. Metaphases show EdU (green), telomeres labelled with TelPNA-C-rich-Cy3 (red) and chromosomes counterstained with DAPI (blue). Scale bar, 10 µm. (E) Quantification of mitotic DNA synthesis at telomeres (single chromatid) in TRF1F/F MEFs infected with shGAPDH control (GFP or CRE), shSMC5 (GFP or CRE) and shPOLD3 (GFP or CRE). Data are represented (n = 50 metaphases) as number of EdU positive telomeres per metaphase with a violin plot, where the median is underlined in red and quartiles in white. One-way ANOVA multiple comparisons (**, p<0.01; ****, p<0.0001) relative to shGAPDH+GFP sample. Source data are provided as a Source Data File.

Figure 6—source data 1. WB and Quantification of SMC5 knock-down efficiency in TRF1F/F MEFs.
Related to Figure 6A and B.
Figure 6—source data 2. POLD3 mRNA levels after KD in TRF1F/F MEFs.
Related to Figure 6C.
Figure 6—source data 3. Quantification of Mitosis DNA synthesis at telomeres in TRF1F/F MEFs with and without POLD3 and SMC5.
Related to Figure 6D and E.

Figure 6.

Figure 6—figure supplement 1. Cell proliferation and EdU incorporation are not affected in TRF1, TRF1-SMC5 and TRF1-POLD3 mutants.

Figure 6—figure supplement 1.

(A) Growth curves showing cell proliferation in TRF1F/F MEFs infected with shGAPDH control (GFP or CRE), shSMC5 (GFP or CRE) and shPOLD3 (GFP or CRE). Population doublings were calculated for each condition. (B) Representative images of IF showing EdU(green) incorporation in MEFs nuclei (DAPI). Quantification of cells (as %) incorporating EdU using IF-staining (n = 250). Cells positive for EdU staining were classified as in S-Phase, while cells negatively stained for EdU were scored as in non-S phase, for the same genetic backgrounds as in A. Source data are provided as a Source Data File.
Figure 6—figure supplement 1—source data 1. Population doublings in TRF1F/F MEFs with and without POLD3 and SMC5.
Related to Figure 6—figure supplement 1A.
Figure 6—figure supplement 1—source data 2. Number of S- and non-S phase in TRF1F/F MEFs with and without POLD3 and SMC5.
Related to Figure 6—figure supplement 1VB.
Figure 6—figure supplement 2. SMC5 and POLD3 are dispensable for TRF1 dependent telomere fragility.

Figure 6—figure supplement 2.

(A) Representative images of metaphases stained with TelPNA-Cy3 probe (red) and DAPI (blue) from TRF1F/F MEFs infected with shGAPDH control (GFP or CRE), shSMC5 (GFP or CRE) and shPOLD3 (GFP or CRE). Scale bar, 10 µm. (B) Enlarged image showing telomere fragility. (C) Quantification of A-B. Data are indicated as % telomere fragility per chromosome. Data from three independent biological replicates are indicated in a violin plot, where the median is underlined in red and quartiles in white. P value, two-tailed student t-test (****, p<0.0001). Source data are provided as a Source Data File.
Figure 6—figure supplement 2—source data 1. Fragile telomeres in TRF1F/F MEFs with and without POLD3 and SMC5.
Related to Figure 6—figure supplement 2C.