53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling (2022)
Figure 5

Figure 5. RASSF1A function at rDNA breaks is not mediated by MST2 kinase.

(A) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RPA and quantified. Representative images are shown in (Fig EV4R). Error bars represent standard deviation and derive from 3 independent experiments.

(B) HeLa cells were pre-treated with the ATMi or ATRi prior to transfection with I-PpoI mRNA. Cells were stained for RAD51 and quantified. Representative images are shown in (Fig EV4S). Error bars represent standard deviation and derive from 3 independent experiments.

(C-F) HeLa cells were treated with siLUCIFERASE (siLUC) or two different siRNAs against RASSF1A (siR1A_1 and siR1A_2) and 48 h later cells were transfected with I-PpoI mRNA to induce rDNA DSBs. 6 h post mRNA transfection, cells were stained for V5 to identify I-PpoI transfected cells and the other indicated antibodies. Representative images (C, E) and quantification of staining (D, F) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.

(G) Western blot analysis from HeLa cell extracts treated with the indicated siRNAs for the indicated antibodies.

(H) Cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for RPA.

(I) Quantification of (H). 107 values were analysed in siLUC and 90 values were analysed in si53BP1.

(J) U2OS cells were treated with siLUCIFERASE (siLUC), siRASSF1A (siR1A) or si53BP1, treated with 10 μM BrdU for 24 h prior to I-PpoI mRNA transfection. 6 h post I-PpoI treated cells were pre-extracted, fixed and stained for BrdU under non denaturing conditions to visualize ssDNA.

(K) Quantification of (J). Fold change of nuclear BrdU signal relative to siLUC is shown. Error bars represent standard deviation and derive from 3 independent experiments.

(L) HeLa cells were transfected with I-PpoI endonuclease and stained for MST2 and γH2AX. Fluorescence intensity profiles of MST2 (green) and γH2AX (red) signals across the HeLa nuclei are shown. Position of line scan indicated by the yellow line.

(M) Validation of the MST2 siRNA with western blot.

(N) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were treated with 5-EU for 30 min prior to fixation and assessed for incorporation.

(O) Quantification of 5-EU intensity in siLUCIFERASE (siLUC) or siMST2 treated cells transfected with I-PpoI mRNA. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values. 100 values were analyzed in each condition.

(P) HeLa cells were treated with siLUCIFERASE (siLUC) or siMST2 and 48 h post transfection cells were transfected with I-PpoI mRNA. 6 h post transfection cells were fixed and stained for RASSF1A (R1A) and γH2AX.

(Q) Quantification of (P). Error bars represent standard deviation and derive from 3 independent experiments.

(R-W) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were stained for ATM-pS1981 (R), KAP1-pS824 (T) and RPA (V) and quantified (S, U, W). 108 values in (S), 95 values in (U) and 100 values in (W) were analysed.

Data information: DNA was stained with DAPI. Scale bars = 10 μm. P values in (A, B, D, F, K and Q) were calculated using two-tailed student t-test. P values in (I, O, S, U and W) were calculated using a Mann-Whitney test. * Iindicates P=0.05-0.01, ** indicates P= 0.01-0.001, *** indicates P ≤ 0.001.