Fig. 2: TCAF1 protects replication forks under stress through the Ca2+-CaMKK2-AMPK-Exo1 signaling pathway. | Nature Communications

Fig. 2: TCAF1 protects replication forks under stress through the Ca2+-CaMKK2-AMPK-Exo1 signaling pathway.

From: TCAF1 promotes TRPV2-mediated Ca2+ release in response to cytosolic DNA to protect stressed replication forks

Fig. 2

A Left panel: siRNA-mediated knockdown of Exo1 in control- and TCAF1-knockdown HeLa cells. Right panel: Effects of Exo1 knockdown on fork resection in control-knockdown and TCAF1-knockdown cells treated with HU (2 mM, 5 h) or H2O. Red bars represent the mean BrdU intensity of BrdU-positive cells. At least 1,000 cells were analyzed for each sample. n = 3, ****, p ≤ 0.0001 (two-tailed, unpaired t-test). See the source data for the exact P values. B Effects of Exo1 knockdown on chromosomal stability in control-knockdown and TCAF1-knockdown HeLa cells after HU (4 mM, 4 h) or H2O treatment. Top panel: Representative images of metaphase chromosome spreads for the indicated samples (scale bar, 25 μm). Chromosomal aberrations are marked by arrows. Bottom panel: Quantified result of the samples depicted in the top panel. 150 metaphases from three independent experiments were examined for each sample. C Effects of Exo1 knockdown on the clonogenic survival of control-knockdown and TCAF1-knockdown HeLa cells treated with indicated concentrations of HU for 24 h. Data represent mean ± S.D. from triplicates. **, p ≤ 0.01 (two-tailed, unpaired t-test). See the source data for the exact P values. D Effects of TCAF1 knockdown on HU-induced ER Ca2+ release in HeLa cells. Left panel: Representative images of the GCaMPer signal (scale bar, 25 μm). Right panel: Quantified GCaMPer signal in S phase-synchronized cells treated with HU (4 mM, 4 h) or H2O. 250 cells were scored for each sample. Black bars represent the mean. n = 3, ****, p ≤ 0.0001 (two-tailed, unpaired t-test). See the source data for the exact P values. E Effects of TCAF1 knockdown on HU-induced iCa2+ elevation in HeLa cells. Left panel: Representative images of the GCaMP6s signal (scale bar, 25 μm). Right panel: Quantified GCaMP6s signal in S phase-synchronized cells treated with HU (4 mM, 4 h) or H2O. 250 cells were scored for each sample. Red bars represent the mean. n = 3, ****, p ≤ 0.0001 (two-tailed, unpaired t-test). See the source data for the exact P values. F Effects of TCAF1 knockdown on HU-induced T172-phosphorylation of AMPKα and S345-phosphorylation of Chk1 in HeLa cells treated with HU (4 mM, 6 h) or H2O. Source data are provided as a Source Data file.

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