Figure S1 from Radiation-Induced Myofibroblasts Promote Tumor Growth via Mitochondrial ROS–Activated TGFβ Signaling

Induction of alpha-SMA after acute single radiation</p

Supplemental Figure Legends from Radiation-Induced Myofibroblasts Promote Tumor Growth via Mitochondrial ROS–Activated TGFβ Signaling

S1. The percentage of TIG-3 and MRC-5 cells with alpha-SMA staining exposed to indicated doses is shown in the graph. S2. Tumor volumes of HeLa, HeLa+MRC-5 0FR tumors, HeLa+MRC-5 0.05FR tumors, HeLa+MRC-5 NAC 0FR tumors, and HeLa+MRC-5 0.05FR tumors.</p

FIgure S2 from Radiation-Induced Myofibroblasts Promote Tumor Growth via Mitochondrial ROS–Activated TGFβ Signaling

Individual tumor measurements data</p

The ATM/TP53/p21 pathway maintains genomic integrity under chronic γ-irradiation conditions by regulating cell-fate decisions.

<p>(A) BJ1/hT cells were transfected with indicated siRNAs and cultured for 4 days under chronic γ-irradiation conditions (0.694 mGy mGy/min). β-gal activity was used to assess cellular senescence. Values represent the mean ± SD of three independent experiments. ***<i>P<0.001</i>. (B) BJ1/hT cells were transfected with indicated siRNAs and then cultured under chronic γ-irradiation conditions at indicated dose rates for 4 days. After an additional 10 days of growth in unirradiated conditions, the number of macroscopic colonies was determined. Values were normalized to control and represent the mean ± SD of three independent experiments. (C) BJ1/hT cells were transfected with indicated siRNAs and then cultured under chronic γ-irradiation conditions at indicated dose rates for 4 days (-d4). Some cells were cultured an additional 4 days under unirradiated conditions (-d4-d8). Values represent the mean ± SD of three independent wells. (D) Wild-type or <i>TP53</i> null mice were exposed to acute (ii, iv, vi) or chronic (iii, v, vii) γ-irradiation treatment conditions, as indicated on the left. β-gal activity was used to assess cellular senescence of primary lung cells isolated from these mice. Values represent the mean ± SD of independent cultures from 2 mice. *<i>P<0.05</i>. (E) BJ1/hT cells were transfected with indicated siRNA and then cultured without (left) or with (right) KU55933 (10 µM) under chronic γ-radiation conditions at indicated dose rates for 4 days. The number of micronuclei was then assessed. Values represent the mean ± SD of at least three independent experiments.</p

The dose rate of chronic γ-irradiation affects cell-fate decisions.

<p><b>(A) Colony-forming ability of fibroblasts following chronic γ-irradiation.</b> The experimental scheme is illustrated to the left. TIG-3 p27 cells (2×10²) were exposed to different dose-rates of γ-irradiation for 10 days and then allowed to grow under unirradiated conditions for an additional 10 days. Representative pictures of crystal violet-stained colonies are shown for each dose rate. (B) Cellular senescence induced by acute or chronic γ-irradiation. BJ1/hT cells (4×10³) were exposed to the treatment condition indicated on the left. Grey arrows indicate chronic γ-irradiation at indicated dose rates. Black arrows indicate acute γ-irradiation of indicated doses. Following 1 day of recovery and 6 days of additional growth, cells were stained for β-gal activity to assess levels of senescence (right). Values represent the mean ± SD of three independent experiments. *<i>P<0.05</i>, ***<i>P<0.001</i>. (C) Cellular senescence induced by acute or chronic γ-irradiation <i>in vivo</i>. Female C57BL/6 mice were exposed to the treatment condition indicated to the left. Grey arrows indicate chronic γ-irradiation at indicated dose rates. Black arrows indicate acute γ-irradiation of indicated doses. Primary cells isolated from lungs of irradiated mice were stained for β-gal activity to assess levels of senescence (right). Values represent the mean ± SD of independent cultures from 2 mice. ***<i>P<0.001</i>.</p

Depleting RAD18 suppressed the response to DNA damage induced by IR.

<p>HEK293 cells were transfected with si-ctrl or si-RAD18, irradiated with 2 or 4 Gy, and then lysed at the indicated time points after irradiation. Samples were analyzed by western blotting with the indicated antibodies.</p

<b>Comparison of colony forming abilities of different types of cells for acute versus chronic γ-irradiation (5 Gy).</b>

<p>Values represent the mean ± SD of three independent experiments.</p>1<p>Survival fractions were determined based on crystal violet staining.</p>2<p>Dose rate of 1.0 Gy/min for 5 min, total dose; 5 Gy. Cells were cultured for 10 days following acute γ-irradiation.</p>3<p>Cells were cultured for 10 days at dose rate of 0.347 mGy/min, total dose; 5 Gy.</p

RAD18 is involved in IR-induced activation of the G2/M phase cell cycle checkpoint.

<p>(A) HT1080 human cancer cells were transfected with si-ctrl or si-RAD18. After 48 hrs, the expression levels of RAD18 in the insoluble fractions were determined by western blotting. (B) Cell proliferation was measured in si-ctrl and si-RAD18 treated cells. Each value represents the mean (+standard deviation) of the results from three independent experiments. (C) Cells were exposed to 4Gy IR, fixed at the indicated time points after irradiation, stained with propidium iodide (PI), and the cell cycle distributions were determined by flow cytometry. (D) Cells were exposed to 1, 2, and 4 Gy IR, and then fixed at 6 hrs after irradiation. Fixed cells were stained with PI and analyzed by flow cytometry.</p

The ATM/TP53/p21 pathway mediates the effect of chronic γ-irradiation on cell proliferation.

<p>(A) Western blotting was used to determine levels of TP53, phosphorylated TP53 (Ser-15), MDM2, MDMX, and p21 in human fibroblasts (TIG-3 p27) cultured under chronic γ-irradiation conditions for 24 or 96 hours (dose rates are indicated). β-actin served as a loading control. (B) Knockdown of TP53 or p21 in BJ1/hT cells abolishes the growth inhibitory effect of chronic γ-irradiation. Western blot analysis of cells transfected with control siRNA (si-Ctrl), TP53-specific siRNA (si-P53), or P21-specific siRNA (si-P21) are shown (top). β-tubulin served as a loading control. Whole cell lysates were prepared 48 hours after the transfection. Forty hours after the transfection. Forty-eight hours after transfection, BJ1/hT cells were placed into culture and grown under unirradiated control conditions (white circles) or a γ-irradiation dose rate of 0.347 mGy mGy/min (grey circles). The number of cells per dish was counted after indicated periods of time. (C) ATM kinase is activated in response to chronic γ-irradiation. BJ1/hT cells were exposed to indicated dose rates of chronic γ-irradiation in the presence or absence of the ATM kinase inhibitor KU55933 (10 µM). ATM kinase activity was assessed by detecting phosphorylated TP53 (Ser15) or phosphorylated CHEK2 (Thr68) by Western blotting. TP53 activation and P21 induction by chronic γ-irradiation were examined by Western blotting. (D) Inhibition of ATM kinase, but not DNA-PKcs, attenuates the growth inhibitory effect of chronic γ-irradiation. BJ1/hT cells were cultured under chronic γ-irradiation conditions at indicated dose rates in the presence or absence of KU55933 (10 µM) or the DNA-PKcs inhibitor NU7026 (10 µM). Values indicate the mean ± SD of three independent wells.</p

Chronic γ-irradiation suppresses the proliferation of human fibroblasts by inducing a G1 cell-cycle arrest.

<p>(A) Dose-dependent effect of chronic γ-irradiation on the proliferation of primary human fibroblasts (TIG-3 p27 cells). Values represent the mean ± SD of three independent wells. (B) Representative cell cycle distribution of TIG-3 p27 cells after 4 days of chronic γ-irradiation. (C) The frequency distribution of DNA damage-associated TP53BP1-foci in TIG-3 p27 (left) or BJ1/hT (right) cells exposed to 4 days of chronic γ-irradiation. At least 1×10³ cells per well were examined to determine the frequency distribution. The size of the bubble is proportional to the number of cells with that number of TP53BP1-foci. Black bars indicate the mean number of TP53BP1-foci per cell. (D) Time-course analysis of TP53BP1 foci in BJ1/hT cell exposed to 0.347 (left) or 0.694 (right) mGy/min of chronic γ-irradiation.</p