Common approaches to count DNA damage foci.

(A) DAPI channel with 5 µm scale bar, (B) γH2AX channel, (C) Composite of A) and B). (D-E) Foci counting with Find Maxima tool in ImageJ with a threshold of D) 600 and E) 300. (F) Foci counting using Imaris Spots with an automatic threshold and an expected spot size of 1 µm. (G) Number of maxima per nucleus from different thresholds using ImageJ for the same cell. (H) Foci counting comparison between a user defined threshold in ImageJ and automatic threshold in Imaris on the same cell. (I) Intensity adjusted γH2AX channel showing lower intensity foci (white arrows), larger foci (green arrows) and higher background intensities (blue arrows).

Counting foci using image correlation spectroscopy correlates with optimized measurements using existing approaches.

(A) Schematic population of monomeric fluorescent particles where emitting particles (green) are in a diffraction limited excitation focus (cyan) – not to scale. (B) Schematic showing increase in particle density from A) which results in an increase in average fluorescence intensity. (C) Schematic showing clustering of fluorescent particles where the total number of independent particle clusters is the same as A). Spatial ICS measures the average number of independent fluorescent units per area which, along with the average fluorescence intensity, is used to calculate the average degree of aggregation of the particles. (D) Intensity-based segmentation of the DAPI channel with an automatic threshold using the MATLAB function imbinarize to create a mask of the nucelus. The mask specifies the ROI for ICS analysis of the γH2AX channel, outputting mean number of independent fluorescent particles per focal spot area (inverse of the intensity normalized spatial correlation function amplitude). (E) and (F) Comparison of the ICS parameter TNoP with E) ImageJ Find Maxima tool with a user defined threshold and F) Imaris Spots with an automatic threshold for the same nuclei. A linear line of best fit is shown on each graph with the resulting R2.

Imaris Spots and ICS capture dose dependent DNA damage through γH2AX foci in SKOV3 cells.

(A-C) SKOV3 cells treated with 0.1 mM MMS; A) DAPI channel with 10 µm scale bar, B) γH2AX channel, C) γH2AX channel with intensity-adjustment. (D-L) Data for normalized intensity, normalized number of spots and normalized ICS DA of γH2AX signal across different MMS (D-F), veliparib (G-I) and olaparib (J-L) treatment concentrations in SKOV3 cells. Shown are individual cells, average for each biological repeat (n=3, squares), and average with standard deviation. Results are normalized to average DMSO. MMS; N=2423-3160, veliparib; N=1818-2922, olaparib; N=1489-2956. * indicates p<0.05, ** indicates p<0.001, *** indicates p<1X10−10 via one-way ANOVA significance testing.

Imaris Spots and ICS DA capture dose dependent DNA damage through γH2AX in OVCA429 cells.

A-C) OVCA429 cells treated with 0.1 mM MMS; A) DAPI channel with 10 µm scale bar, B) γH2AX channel, C) γH2AX channel with intensity-adjustment. (D-L) Data for normalized intensity, normalized number of spots and normalized ICS DA of γH2AX signal across different MMS (D-F), veliparib (G-I) and olaparib (J-L) treatment concentrations in OVCA429 cells. Shown are individual cells, average for each biological repeat (n=3, squares), and average with standard deviation. Results are normalized to average DMSO. MMS; N=1194-2068, veliparib; N=1256-1997, olaparib; N=1445-2334. * indicates p<0.05, ** indicates p<0.001, *** indicates p<1X10−10 via one-way ANOVA significance testing.

Dose-dependent DNA damage impact on RPA1 and RAD51.

(A-C) Control SKOV3 cells, (D-F) SKOV3 cells treated with 1 mM MMS; (A&D) DAPI channel with 10 µm scale bar, (B&E) RPA1 channel, (C&F) RPA1 channel with intensity-adjustment. Normalized RPA1 Imaris Spots (G) and normalized ICS DA (H) in SKOV3 and OVCA429 cells (I&J). Shown are individual cells, average for each biological repeat (n=3, squares), and average with standard deviation. Results are normalized to average DMSO. N=1600-1872 for SKOV3 and N=1320-2198 for OVCA429. (K-M) Control SKOV3 cells, (N-P) SKOV3 cells treated with 1 mM MMS; (K&N) DAPI channel with 10 µm scale bar, (L&O) RAD51 channel, (M&P) RAD51 channel with intensity-adjustment. Normalized RAD51 Imaris Spots (Q) and normalized ICS DA (R) in SKOV3 and OVCA429 cells (S&T). Shown are individual cells, average for each biological repeat (n=3, squares), and average with standard deviation. Results are normalized to average DMSO. N=1597-2241 for SKOV3 and N=1822-2311 for OVCA429. All results, * indicates p<0.05, ** indicates p<0.001, *** indicates p<1X10−10 via one-way ANOVA significance testing.

RPA1 recruitment correlates with cell sensitivity to PARPi.

(A) RPA1 DA as a function of olaparib concentration for HT1080 and HCC1937 cells, shown are average with SEM, n >= 504 cells, 3 biological repeats, and linear fit. (B) RPA1 DA in cells treated with 1 μM PARPi overnight, shown are average, normalized to untreated control, with SEM, n >= 350 cells, 2 biological repeats. (C) RPA1 DA as a function of cell line IC50 for each cell line and PARPi shown in Fig. 5b. Shown are average, normalized to untreated control, with SEM, n >= 350 cells, 2 biological repeats and linear fit (prism) for each cell line. (D) The linear fit slope of RPA DA vs. cell line IC50 as a function of average PARPi z-score for each cell line. Shown are slope fit with SEM and average z-score over three PARPi with st. dev. (E) Western blot of RPA1 expression levels for each cell line, with GAPDH loading control. (F) Normalized protein expression level in each cell line as a function of PARPi z score. Shown are average z-score over three PARPi with st. dev.

Incorporating and validating cell cycle data.

(A) Histograms of DAPI intensities for cells treated with palbociclib, camptothecin or etoposide, 1 μM overnight. (B) Representative assignment of cell cycle based on DAPI histogram (grey), using the first peak as G1 cells. Normalized PCNA intensity (C) and DA (D) in SKOV3 cells that had been classified by DAPI intensity into phases of the cell cycle. Normalized γH2AX intensity (E) and DA (F) in SKOV3 cells that had been classified by DAPI intensity into phases of the cell cycle. Shown are average n>200 cells per classification and standard deviation.

Cell cycle analysis of DDR ICS.

Normalized γH2AX DA and intensity of SKOV3 cells treated with DMSO or olaparib (A), bleomycin (B) or etoposide (C) at 3 different doses (in μM) for 24 hours and separated by cell cycle stage based on DAPI intensity. Data were normalized to cell cycle independent DMSO average. Also shown are the ratios of the number of cells in G2 to G1 for each treatment condition. Data are n=7 biological repeats (box = average) with single cell values and standard deviation, n>200 cells per condition. For clarity all data are significantly different, p<0.05 Student’s t test from the next lower concentration unless marked – ns = no significance, dec = significant decrease.