Fig 1: Fa27 binds TLR4 and reduces LPS stimulation. (A) Fa27 inhibits TLR4 activation by LPS. Scheme of experimental analysis of TLR4 signaling and results obtained in HEK-Blue hTLR4 cells pre-treated for 2 h with different concentrations of Fa27 followed by additional treatment with 10 ng/ml LPS for 10 h. TLR4 stimulation led to a release of secreted embryonic alkaline phosphatase (SEAP) detected at OD 655 nm. LPS treatment increased SEAP release which was reduced by Fa27 pre-treatment. Mean OD values of LPS-treated controls without peptide were set to 100% in each experiment. Data (n=12 obtained in two experiments) are shown as mean +SEM and statistics were calculated relative to LPS stimulation without peptide (*p < 0.05; **p < 0.01; ****p < 0.0001). (B) TLR4 and Fa27 interaction. HEK-Blue hTLR4 cells were seeded 72 h before the experiment and treated for 2 h with N-terminally tagged Flag-Fa27, C-terminally tagged Fa27-Flag or water (ctrl). PLA analysis showed an interaction between both tagged Fa27 peptides and the TLR4. Two positive controls are shown: PLA for TLR4 and its binding partner MD-2 on the cellular surface and for Ku70-Ku80 complexes in the nucleus. The negative control was done with PLA substrates but without antibodies. Margins of cells are marked by white stippled lines and the scale bar indicates 10 µM (n=187-425 from two independent experiments each; mean +SEM; ****p < 0.0001). See also Figure S6 .
Fig 2: BCDX2 paralog complex, but not CX3, promotes replication fork slowing and reversal upon mild CPT treatment.a Rationale for focusing on RAD51C, RAD51D, and XRCC3 downregulation, to perform functional studies on BCDX2 and CX3 complexes during replication stress. Top: schematic of the two main RAD51 paralog complexes. Bottom: differential effects of RAD51C, RAD51D, and XRCC3 inactivation on paralog complex stability/function. b Western blot analysis of RAD51 paralogs upon 48 h downregulation with two different siRNAs. Ctrl, control siRNA; Tubulin, loading control. asterisk, specific band. In b and d multiple gels/blots were processed in parallel, ensuring equal and comparable loading across gels. The experiment was performed twice yielding similar results. c DNA fiber analysis of U2OS cells 48 h after transfection with a control siRNA (siCtrl) or with siRNAs targeting RAD51C, RAD51D, and XRCC3. Top-left: schematic of the CldU/IdU pulse-labeling protocol used to evaluate fork progression upon 50 nM CPT treatment. Bottom-left: representative DNA fibers from each genetic condition. Left: IdU/CIdU ratio is plotted as a readout of fork progression. In c and e, the numbers indicate the mean value; a minimum of 150 forks was scored in two independent experiments yielding similar results. Bounds of box are 25–75th percentile, center shows the median, whiskers indicate the 10–90 percentiles, data points outside this range are drawn as individual dots. Statistical analysis: Kruskal–Wallis test; ns not significant; ****p-value < 0.0001, ***p-value = 0.0003. d Western blot analysis of RAD51 paralogs in Knock-Out (KO) U2OS cells and in cells reconstituted of the respective protein (+). KU70, loading control. The experiment was performed twice yielding similar results. e DNA fiber analysis of KO and reconstituted (+) U2OS cells labeled as in c. f, g Frequency of reversed replication forks in U2OS cells transfected with control siRNA (Ctrl) or with siRNAs targeting RAD51C, RAD51D, or XRCC3 for 48 h and treated with 50 nM CPT (1 h). f Electron micrograph of a representative reversed replication fork from CPT-treated U2OS cells (P parental duplex, D daughter duplexes, R regressed arm; scale-bar, 100 nm). g Graph-bar showing mean and SD from three independent EM experiments. In brackets, total number of molecules analyzed per condition. Statistical analysis: one-way ANOVA followed by Bonferroni test; ns not significant; ***p-value = 0.0004. Source data for b–g are provided in the Source Data file.
Fig 3: CX3 complex promotes reversed fork restart in U2OS cells.a DNA fiber analysis of U2OS cells transfected for 24–48 h (24 h for RAD51, 48 h for all the other genes) with a control siRNA (Ctrl) or with siRNAs targeting RAD51, RAD51C, RAD51D, and XRCC3 to investigate replication fork restart upon HU. Top: schematic CldU/IdU pulse-labeling protocol to evaluate fork restart upon HU treatment (2 mM, 2 h). Bottom-left: representative stalled and restarting forks. Bottom-right: frequency of fork stalling for each genetic condition. In a–d, the graph-bar depicts mean and SD from three independent experiments. Statistical analysis: one-way ANOVA followed by Bonferroni test; ns not significant; ****p-value < 0.0001. b DNA fiber analysis of U2OS cells double transfected with a control siRNA (Ctrl) or with a siRNA targeting XRCC3 (48 h) or RAD51 (24 h) to investigate replication fork restart upon HU treatments; labeling protocol as in a. Top: levels of indicated proteins, assessed by western blot; Tubulin, loading control. In b and d multiple gels/blots were processed in parallel, ensuring equal and comparable loading across gels. Bottom: frequency of fork stalling. ns not significant; **p-value = 0.0068 (1 versus 3) and 0.0087 (3 versus 4). c DNA fiber analysis of wild-type (WT) or ZRANB3 Knock-Out (KO) U2OS cells transfected with a control siRNA (Ctrl) or with siRNAs targeting XRCC3 (48 h) to investigate replication fork restart upon HU treatments; labeling protocol as in a. Top: indicated protein levels, assessed by western blot; Tubulin, loading control; asterisk, specific band. Bottom: frequency of fork stalling. ns not significant; ****p-value < 0.001. d DNA fiber analysis of U2OS cells double transfected with a control siRNA (Ctrl), with siRNAs targeting XRCC3 (48 h) or RAD51D (60 h) to investigate replication fork restart upon HU treatments; labeling protocol as in a. Top: indicated protein levels, assessed by western blot; KU70, loading control. Bottom: frequency of fork stalling. ns not significant; **p-value = 0.0024; ***p-value = 0.0007. Source data for a–d are provided in the Source Data file.
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