53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling (2022)
Figure 4

Figure 4. RASSF1A depletion results in impaired rDNA damage response.

(A) U2OS cells were transfected with either HA-53BP1WT (aa1-1972), HA-53BP1ΔBRCT (aa 1-1709), HA-53BP1ΔΝterminus (aa 921-1972) or empty pcDNA3 vector and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with an HA tag antibody. Western blot analysis of total cell extracts and the IPs is shown.

(B) U2OS cells were transfected with either full length MYC-RASSF1A or with the indicated RASSF1A deletion mutants and 24 h later transfected with I-PpoI mRNA. Cell lysates were subjected to immunoprecipitation with a MYC-tag antibody. Western blot analysis of the total cell extracts and the IPs is shown.

(C) HeLa cells were treated with siLUCIFERASE (siLUC) or si53BP1 and 48 h later transfected with I-PpoI mRNA and stained for ATM-pS1981.

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

(E-H) 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 (E, F) and quantification of staining (G, H) for the indicated antibodies are shown. Error bars represent standard deviation and derive from 3 independent experiments.

(I) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against UBF and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.

(J) HeLa cells were treated with siLUCIFERASE (siLUC), siRASSF1A or si53BP1 and 48 h later transfected with the I-PpoI mRNA. Cells were fixed and underwent ImmunoFISH against ATM-pS1981 and an rDNA probe. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines.

(K) HeLa cells were treated with siLUCIFERASE (siLUC) or siRASSF1A followed by I-PpoI mRNA transfection. 6 h later cells were stained for 53BP1.

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

(M) U2OS cells were transfected with MYC-RASSF1A (1-340) or a RASSF1A mutant that lacks the SARAH domain, MYC-RASSF1A (1-288). Cells were transfected with I-PpoI mRNA and stained with the indicated antibodies.

(N) U2OS cells were transfected with MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and harvested after 6 h. Cell lysates underwent cell fractionation and the chromatin bound fraction was analyzed by western blot.

(O) U2OS cells were treated with siRNA against RASSF1A followed by transfection with MYC-tag, MYC-RASSF1A (1-340) or MYC-RASSF1A (1-288). 24 h later cells were transfected with I-PpoI mRNA and stained for MYC expression and ATM-pS1981.

(P) Quantification of cells with ATM-pS1981 positive nucleolar caps in (O). Error bars represent standard deviation and derive from 3 independent experiments.

Data information: DNA was stained with DAPI. Scale bars = 10 μm. P values in (D, G, H, L and P) were calculated using two-tailed Student 's t-test. * Indicates P=0.05-0.01, ** indicates P= 0.01-0.001, *** indicates P ≤ 0.001