Journal: Scientific Reports

Article Title: RECQL4 is not critical for firing of human DNA replication origins

doi: 10.1038/s41598-024-58404-0

Figure Lengend Snippet: | RECQL4 is not critical for entry into S phase. ( a ) Immunoblot showing depletion of RECQL4 by specific siRNA (si) with PCNA serving as a loading control (top) and flow cytometry analysis of EdU incorporation in asynchronous cells following depletion of TRESLIN or RECQL4 (bottom). Ctrl, control; 2C and 4C, genomic DNA content. ( b ) Graphical representation of the human RECQL4 protein showing its three major protein domains and the sites targeted for mutagenesis. Red and green arrows, sites targeted in the KO and HD clones, respectively. fsH and fsN, frameshift mutations in the Helicase and N-terminal domains, respectively; KM, lysine to methionine substitution in the helicase domain. ( c ) Immunoblot analysis showing the levels of RECQL4 expression in the KO and HD clones and induction of Cyclin E. PCNA and Lamin A serve as loading controls. NE, normal levels of Cyclin E; OE, overexpression of Cyclin E. ( d ) Experimental setup (top) and kinetics of S-phase entry of parental cells and RECQL4 -mutant clones, as ascertained by flow cytometry-based analysis of EdU-positive cells (% EdU +) at different time points after mitotic shake-off (bottom). Averages and standard deviations from three independent experiments are shown. Clone fsH2 entered S phase faster than the parental cells, when Cyclin E was overexpressed ( P < 0.001).

Article Snippet: Antibodies specific for RECQL4 (SDIX, Cat. No. 2547.00.02; Novus Biologicals, Cat. No. NBP2-47,310), Cyclin E (DBS, Cat. No. Mob181), PCNA (Genetex, Cat. No. GTX-20029), Treslin (Abcam, Cat. No. ab124268) and Lamin A (Abcam, Cat. No. ab26300) were used at the concentration recommended by the manufacturer.

Techniques: Western Blot, Control, Flow Cytometry, Mutagenesis, Clone Assay, Expressing, Over Expression

Journal: Scientific Reports

Article Title: RECQL4 is not critical for firing of human DNA replication origins

doi: 10.1038/s41598-024-58404-0

Figure Lengend Snippet: | Targeting RECQL4 has no effect on DNA replication origin firing profiles. ( a ) Experimental setup for EdU-seq. ( b ) and ( d ) Overlay of DNA replication origin firing profiles of parental cells (WT) and RECQL4 -mutant clones for a representative genomic region under conditions of normal Cyclin E expression (NE) ( b ) or Cyclin E overexpression (OE) ( d ). The EdU-seq data are presented as sigma (σ) values. RT; replication timing (blue, early; green, mid S phase); Ge, genes (green, forward direction of transcription; red, reverse; yellow, unspecified; blue, multiple genes within bin); iG, intergenic regions (gray). Bin resolution: 10 kb. ( c ) and ( e ) Correlation plots of the sigma values of all constitutive (CN, purple) and oncogene-induced (Oi, red) origins of the parental cells versus the RECQL4 -mutant clones under conditions of normal Cyclin E expression ( c ) or Cyclin E overexpression ( e ).

Article Snippet: Antibodies specific for RECQL4 (SDIX, Cat. No. 2547.00.02; Novus Biologicals, Cat. No. NBP2-47,310), Cyclin E (DBS, Cat. No. Mob181), PCNA (Genetex, Cat. No. GTX-20029), Treslin (Abcam, Cat. No. ab124268) and Lamin A (Abcam, Cat. No. ab26300) were used at the concentration recommended by the manufacturer.

Techniques: Mutagenesis, Clone Assay, Expressing, Over Expression

Journal: Scientific Reports

Article Title: RECQL4 is not critical for firing of human DNA replication origins

doi: 10.1038/s41598-024-58404-0

Figure Lengend Snippet: | RECQL4 affects DNA replication fork progression. ( a ) Experimental outline for the analysis of fork progression by DNA combing in synchronized cells. ( b ) and ( c ) Lengths of IdU tracks of parental cells (WT) and RECQL4 -mutant clones treated as shown in ( a ). NE, normal levels of Cyclin E ( b ); OE, Cyclin E overexpression ( c ). More than 190 IdU tracks were measured per sample. ( d ) Experimental outline for the analysis of fork progression by DNA combing in asynchronous cells. ( e ) and ( f ) Lengths of CldU ( e ) and IdU ( f ) tracks of parental cells (WT) and RECQL4 -mutant clones treated according to the outline shown in ( d ). The cells expressed normal levels of Cyclin E (NE). More than 150 CldU-IdU double-labeled fibers were measured per sample. For all samples, the median and upper and lower quartiles are indicated by horizontal lines. P values were calculated by a two-way ANOVA with Fisher’s Least Significance Difference test. *, P < 0.05; ****, P < 0.0001; ns: not significant. ( g ) Experimental outline for the study of fork progression by EdU-seq. For the 0 min (’ or min) timepoint, after mitotic shake-off, the cells were incubated with HU and EdU for 14 h. ( h ) Genome-wide averages of the EdU-seq sigma values of the indicated samples and timepoints over genomic regions spanning 0.6 Mb around origins of replication. NE, normal levels of Cyclin E; OE, overexpression of Cyclin E; aσ, adjusted sigma values relative to the no-release (0 min) samples.

Article Snippet: Antibodies specific for RECQL4 (SDIX, Cat. No. 2547.00.02; Novus Biologicals, Cat. No. NBP2-47,310), Cyclin E (DBS, Cat. No. Mob181), PCNA (Genetex, Cat. No. GTX-20029), Treslin (Abcam, Cat. No. ab124268) and Lamin A (Abcam, Cat. No. ab26300) were used at the concentration recommended by the manufacturer.

Techniques: Mutagenesis, Clone Assay, Over Expression, Labeling, Incubation, Genome Wide

Journal: Scientific Reports

Article Title: RECQL4 is not critical for firing of human DNA replication origins

doi: 10.1038/s41598-024-58404-0

Figure Lengend Snippet: | RECQL4 is not critical for MiDAS. ( a ) Experimental outline for the assessment of the presence of MiDAS in mitotic parental (WT) and RECQL4 mutant cells by epifluorescence microscopy. ( b ) Fraction of EdU + mitotic cells, according to ( a ); at least two EdU foci per mitosis were required to consider a cell positive for MiDAS. Means and standard deviation of three independent experiments are shown on the graph. At least 130 mitotic cells were analyzed for each condition. Statistical analysis was performed with 2-way ANOVA with Tukey’s Multiple Comparisons test. ns, not significant. ( c ) Experimental outline for the study of mitotic DNA synthesis by MiDAS-seq. ( d ) Average MiDAS-seq signal (σ values) of all MiDAS regions and heatmap of the MiDAS-seq signal of each MiDAS region ranked according to its genomic size for single- or double-peak regions. Bin resolution: 10 kb. ( e ) Experimental outline for monitoring 53BP1 nuclear bodies in G1. ( f ) Fraction of cells containing 53BP1 nuclear bodies in early G1, according to ( e ). The graph shows averages and standard deviations from three independent experiments, with at least 230 cells analyzed per cell line. No significant differences between the parental cells and RECQL4 mutant clones were observed (2-way ANOVA with Tukey’s Multiple Comparisons test). Aph, aphidicolin; RO, RO3306.

Article Snippet: Antibodies specific for RECQL4 (SDIX, Cat. No. 2547.00.02; Novus Biologicals, Cat. No. NBP2-47,310), Cyclin E (DBS, Cat. No. Mob181), PCNA (Genetex, Cat. No. GTX-20029), Treslin (Abcam, Cat. No. ab124268) and Lamin A (Abcam, Cat. No. ab26300) were used at the concentration recommended by the manufacturer.

Techniques: Mutagenesis, Epifluorescence Microscopy, Standard Deviation, DNA Synthesis, Clone Assay