Fig. 3: TFIP11 accumulates at stalled forks.
From: TFIP11 promotes replication fork reversal to preserve genome stability

A Input and iPOND samples were analyzed by Western blotting. HeLa cells were labeled with 10 μM EdU for 15 min followed by a 1 h chase with 10 μM thymidine (Thd). The cells were then challenged with 4 mM HU for 3 h prior to crosslinking with 1% formaldehyde. No Clk, no-click samples; rxn, reaction. B Schematic diagram of the PLA. C, D HeLa cells expressing empty vector or HA-Flag-tagged TFIP11 were labeled with 10 μM EdU for 15 min before treatment with 4 mM HU for 3 h. Click chemistry was then used to conjugate biotin to EdU. Representative images of PLA foci (red) shown in (C). Scale bar, 10 μm. Quantification of the average number of PLA foci per focus-positive cell or the percentage of cells with PLA foci (D). Data represent means ± SD from three independent experiments. From left, n = 168, 168, 168, 168 cells. Statistical analysis was calculated with two-tailed, unpaired t-test. E Strategy for generation of SF-TFIP11 knock-in HeLa cell line. F The cell lysates were immunoprecipitated with S protein beads and were probed with anti-Flag or anti-TFIP11 antibody. G–I SF-TFIP11 knock-in HeLa cells were labeled with 10 μM EdU for 15 min before treatment with 4 mM HU for 3 h. PLA was conducted with anti-Flag and anti-biotin antibodies. Representative images of PLA foci (red) shown in (G). Scale bar, 10 μm. Quantification of the average number of PLA foci per focus-positive cell or the percentage of cells with PLA foci (H). Data represent means ± SD from three independent experiments. From left, n = 104, 100, 105, 322, 348, 370 cells. P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. Knockdown efficiency of TFIP11 was confirmed (I). Source data are provided as a Source Data file.