Presentation on theme: "AB Supplementary Figure S1. (A) H1299 cells harboring pGC were transfected with I-SceI-expressing vector. Percentage of GFP + cells was assessed after."— Presentation transcript:
AB Supplementary Figure S1. (A) H1299 cells harboring pGC were transfected with I-SceI-expressing vector. Percentage of GFP + cells was assessed after inhibition of ATM (10µM KU55933), MRE11 (25µM Mirin) or both 48h post-transfection as an indication for HR efficiency. (B) inhibition of MRE11 does not affect ATM signaling. A549 cells were treated with 25µM mirin for 2h prior to irradiation with 3Gy and after 30 min phosphorylation of both ATM at S1981 and CHK1 at Thr 68 were investigated. (C) Western blot analysis showing efficient siRNA–mediated knockdown of both ATM (left panel) and MRE11 (right panel). C
Supplementary Figure S2. After irradiation with 2Gy, RAD51 colocalized with γH2AX foci were monitored in A549 cells after ATM knockdown (siATM), MRE11 (siMRE11) or both (siATM/siMRE11). Knockdown of ATM or MRE11 should a significant reduction in RAD51 foci at 3h time point after 2Gy. Knockdown of both proteins did not show any synergistic effect on the number of RAD51 foci at this time point. At the 24h time point, the number of residual RAD51 foci was significantly higher upon ATM knockdown than after MRE11 knockdown. Simultaneous depletion of both proteins showed a slight increase in RAD51 compared to ATM depeltion alone. In all cases, the number of foci measured in non-irradiated cells was subtracted from that observed in irradiated cells. Error bars represent the SEM of three independent experiments.
Supplementary Figure S3. Radiosensitivity of exponentially growing A549 cells was measured by colony forming assay after inhibition of ATM (10µM KU55933) and/or MRE11 (25µM Mirin) either 2h (2h-post) or 4h (4-post) after the indicated X-ray doses. Error bars represent the SEM of three independent experiments.
AB Supplementary Figure S4. (A) Representative IF photos for RAD51 foci (Red) in CenpF + S/G2 (green) A549 cells at the indicated time points after ATM inhibition either 1h prior to (-pre) or 2h after (-post) 2Gy. 5µM Aphidicoline (APH) was added to block the transition of S-phase cells into G2. (B) Quantification of RAD51 foci in at least 50 CenpF + cells for each indicated time point. In all cases the number of foci measured in non- irradiated cells was subtracted from the number of IRIF observed in irradiated cells. Error bars represent the SEM of three independent experiments.
Supplementary Figure S5. (A) Identification of cells in different cell ‐ cycle phases in A549 cells. Cells were pulsed labeled with EdU and co-stained with CenpF. Using these two markers the different cell cycle phases are distinguished (G1-phase cells: CenpF-negative, S-phase cells: CenpF-positive and EdU-positive, G2-phase cells: CenpF-positive and EdU-negative). (B) Examples of discrete RAD51 foci in CenpF + and EdU - G2 cells (left panel) or CenpF + and EdU + S-phase cells (right panel) at the indicated time points after 2 Gy of X-ray.
Supplementary Figure S6. (A) Western blot showing an efficient KAP1 depletion using siRNA (+siKAP1) in A549 cells. (B) ATM was then inhibited in A549 cells either 1h prior to (ATMi-pre) or 2h after (ATMi-post) 2Gy and RAD51 foci (relative to mock irradiated cells) were monitored in CenpF + S/G2 cells at the indicated time points. Non-specific siRNA was used as a control (- siKAP1). In all cases the number of foci measured in non-irradiated cells was subtracted from the number of IRIF observed in irradiated cells. Error bars represent the SEM of three independent experiments. A B
Supplementary Figure S7. Detection of sister chromatid exchanges after inhibition of ATM either prior to or after 2Gy. A549 cells were treated with or without ATMi prior to or after 2Gy then the cells were incubated overnight with colcemid (added at 5 h after irradiation) to collect cells in mitosis. (A) Representative example of a metaphase spread shows multiple SCE (indicated by arrows). (B) quantification of SCE per metaphase spread in un-irradiated (UT) or irradiated (2Gy) cells. At least 40 metaphases per data point were analyzed. AB
Supplementary Figure S8. Detection of apoptosis in via caspase activity. ATM was inhibited in A549 cells either prior to (pr-IR) or 2h after (post-IR) X-irradiation with 2 Gy. Caspase activity (as indicator for apoptosis) was then analyzed at 24h time point. Values obtained for non-irradiated cells were subtracted. A549 or Jurkat cells were irradiated with 2 Gy and incubated with 1 μM staurosporine for 24 h were used as a positive control.
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