Table 7.1 Screen candidates based on overall γH2AX levels.
Screen candidates were defined as genes whose knockdown led to a robust z-score < -2 or > 2 of overall γH2AX levels with at least two of three siRNAs. All candidates were sorted by their achieved cumulative robust z-scores in γH2AX levels. Column A to C indicate the respective siRNA of the library. Robust z-scores of the candidates in EdU-positive cells and their influence on EdU incorporation are coated blue and green, respectively.
Candidates which were further validated are marked red. Candidates achieving a robust-Z-score < -2 or > 2 in EdU-positive cells are marked blue.
γH2AX in all cells γH2AX in EdU-positive cells EdU incorporation Gene
Symbol
Gene Name robust z-score robust z-score robust z-score
A B C Cum. A B C Cum. A B C Cum.
oncogene homolog
Table 7.2 Screen candidates based on γH2AX levels in EdU-positive cells.
Screen candidates were defined as genes whose knockdown led to a robust z-score < -2 or > 2 of γH2AX levels in EdU-positive cells with at least two of three siRNAs. All candidates (coated blue) were sorted by their achieved cumulative robust z-score in EdU-gated γH2AX levels. Column A to C indicate the respective siRNA of the library. Robust z-scores of the candidates in all cells and their influence on EdU incorporation are coated grey and green, respectively. Candidates which have been further validated are marked red. Candidates achieving a robust z-score < -2 or > 2 in overall γH2AX levels are marked blue.
γH2AX in EdU-positive cells γH2AX in all cells EdU incorporation
Gene Symbol
Gene Name
robust z-score robust z-score robust z-score
A B C Cum. A B C Cum. A B C Cum.
CIB1
BAZ2A
Bromodomain adjacent to zinc finger domain,
2A 0,40 6,40 2,38 9,17 0,63 0,87 0,85 2,34 -0,96 -1,42 8,57 6,19
RNF25 Ring finger protein 25 0,56 2,32 6,49 9,37 -1,19 -4,47 2,71 -2,95 -0,84 -1,27 -0,30 -2,41 RPA1 Replication protein A1 2,64 5,18 2,55 10,37 2,50 1,87 1,80 6,17 2,70 -0,41 3,94 6,22
POLD1
Polymerase (DNA directed), delta 1, catalytic subunit
125kDa 5,34 3,79 2,00 11,13 -4,06 -1,27 -0,48 -5,81 -1,93 -1,65 -0,88 -4,45
SRCAP
Snf2-related CREBBP
activator protein 7,26 1,56 2,33 11,15 0,06 1,08 -0,86 0,28 -1,25 1,20 -1,81 -1,87
CHD8
Chromodomain helicase DNA binding
protein 8 6,99 1,66 3,79 12,44 7,49 1,70 3,46 12,65 15,65 2,74 0,72 19,11
RFC1
Replication factor C
(activator 1) 1, 145kDa 17,72 2,76 1,78 22,26 1,07 3,60 1,57 6,24 -1,97 5,71 2,36 6,09
CHEK1
CHK1 checkpoint
homolog (S. pombe) 13,76 10,58 0,87 25,21 11,91 6,97 0,83 19,71 1,35 -0,61 -0,99 -0,24
Table 7.3 Relevant biological processes associated with screen candidates.
All gene products whose knockdown caused a significant change in γH2AX levels, i.e. a robust z-score >2 of at least 2 of 3 siRNAs (see table 7.1 and 7.2) were annotated to functional categories using the DAVID functional annotation tool for gene ontology of biological processes. A selection of biological processes which are relevant for the cell’s reaction to chemotherapy is displayed in the table, corresponding to figure 5.11.
Biological process Candidates whose knockdown causes decreased γH2AX levels DNA repair RAD23B, HUS1, SMUG1, MDC1, CCNO,
FANCA, ATRIP, CIB1, C9ORF102,
ds-break repair UBE2N, RNF8, HUS1, CIB1 RPA1, VCP, SOD1
BE repair SMUG1, CCNO POLD1, PCNA, APEX1, PARP2
NE repair RPA1, POLD4, RFC3, RFC1, POLD1,
PCNA HELLS, CIB1, C9ORF102, UBE2N, RNF8, RUVBL2,
DNA replication HUS1, TERF2IP ATR, PCNA, POLD1, POLD4, RFC1,
RFC3, RPA1, RPA4, RPAIN
apoptosis HELLS, MYC CHD8, PHB, SOD1, TOP2A, VCP
cell cycle ATRIP, CENPH, FANCA, HELLS, HUS1, MDC1, MYC, REC8, RNF8, SUV39H2 chromatin remodeling ARID1A, HDAC5, HELLS, SUV39H2 BAZ2A, CHD8
chromatin assembly HELLS, SUV39H2 CHD8, HIST1H1C, H1FX, SHPRH
DNA methylation HELLS BAZ2A
histone deacetylation HDAC5 BAZ2A, PHB histone ubiquitination RNF8, UBE2N
regulation of transcription
ARID1A, HDAC5, HELLS, MYC, RUVBL2, SOX15, SUV39H2, TERF2IP, UBE2N
BAZ2A, CHD8, CHMP1A, GTF2H4, PHB, RFC1, SRCAP, ZNFX1
Figure 7.1 Results of RNAi-based screening for EdU levels as measure for DNA replication.
Supplementary data
corresponding to Figure 4.6. All target genes where ranked according to the cumulative robust z-score of EdU incorporation. The 20 target genes with the highest and lowest cumulative z-scores are shown in magnification.
PC=positive control
Figure 7.2 Control-based validation of main hits in EdU-positive cells.
PANC-1 cells were transfected with siRNAs as indicated and (a) left untreated or (b) exposed to 300 nM gemcitabine for 22 h. Cells were then fixed and stained for immunofluorescence analysis. H2AX phosphorylation was quantified in EdU-positive cells. All candidates were ranked according to cumulative relative changes in γH2AX compared to control knockdown resulting from three different siRNA sequences for every target gene. The relative γH2AX induction by each siRNA is shown for every target gene.
Figure 7.3 Relative changes in EdU levels caused by main hits of the screen.
PANC-1 cells were transfected with siRNAs as indicated and (a) left untreated or (b) exposed to 300 nM gemcitabine for 22 h. Cells were then fixed and stained for immunofluorescence analysis. EdU incorporation was quantified and compared to control transfection. All candidates were ranked according to cumulative relative changes in EdU levels compared to control knockdown resulting from three different siRNA sequences for every target gene. The relative γH2AX induction by each siRNA is shown for every target gene.
Figure 7.4 Control-based validation of selected hits in MIA PaCa-2 cells.
Genes whose depletion proved to have influence on H2AX phosphorylation in PANC-1 cells and which were of interest as novel DNA damage mediators were selected and tested in MIA PaCa-2 cells. Cells were transfected with siRNAs as indicated and either left untreated (left column) or exposed to 300 nM gemcitabine for 22 h (right column). Cells were fixed and stained for immunofluorescence analysis and EdU as well as γH2AX levels were quantified. Relative changes in (a) overall γH2AX levels, (b) γH2AX levels in EdU-positive cells and (c) incorporation of EdU are ranked according to the cumulative changes of all three siRNAs against the respective target gene. The relative change of every readout is displayed for each siRNA individually.
Figure 7.5 Control-based validation of selected hits in PaTu8902 cells.
Genes whose depletion proved to have influence on H2AX phosphorylation in PANC-1 cells and which were of interest as novel DNA damage mediators were selected and tested in PaTu8902 cells. Cells were transfected with siRNAs as indicated and either left untreated (left column) or exposed to 300 nM gemcitabine for 22 h (right column). Cells were then fixed and stained for immunofluorescence analysis and EdU as well as γH2AX levels were quantified. Relative changes in (a) overall γH2AX levels, (b) γH2AX levels in EdU-positive cells and (c) incorporation of EdU are ranked according to the cumulative changes of all three siRNAs against the respective target gene. The relative change of every readout is displayed for each siRNA individually.
Figure 7.6Control-based validation of selected hits in U2OS cells.
Genes whose depletion proved to have influence on H2AX phosphorylation in PANC-1 cells and which were of interest as novel DNA damage mediators were selected and tested in U2OS cells. Cells were transfected with siRNAs as indicated and either left untreated (left column) or exposed to 300 nM gemcitabine for 22 h (right column). Cells were fixed and stained for immunofluorescence analysis and EdU as well as γH2AX levels were quantified. Relative changes in (a) overall γH2AX levels, (b) γH2AX levels in EdU-positive cells and (c) incorporation of EdU are ranked according to the cumulative changes of all three siRNAs against the respective target gene. The relative change of every readout is displayed for each siRNA individually.
Figure 7.7 Effect of candidate gene knockdown on γH2AX is not attributable to a decrease in Rad51 or p53 levels.
PANC-1 cells were transfected with the indicated siRNAs and all wells were treated with 300 nM gemcitabine for 22 h according to screening conditions.
Cell lysates were analyzed by immuno- blotting.