Journal: bioRxiv

Article Title: SETD2 safeguards the genome against isochromosome formation

doi: 10.1101/2022.10.25.513694

Figure Lengend Snippet: a, Images of wild-type, heterozygous or homozygous deletion of Setd2 have lagging (blue arrows) and bridging (orange arrows) chromosomes during anaphase. MEFs with no, one or two floxed alleles of Setd2 ( Wild-type/Wt , Flox/Wt , Flox/Flox , respectively) were either treated with Vehicle (EtOH, control in top panels) or 4-OHT (excise floxed alleles of Setd2 , bottom panels) for three days, fixed and counterstained with Hoechst (grey). Scale bars are 5μm. b, Quantification of chromosome segregation errors during anaphase and early telophase in control (vehicle treated) or 4-OHT treated MEFs described in a . n=198 wt/wt vehicle, n=215 wt/wt 4-OHT, n=298 fl/wt vehicle, n=227 fl/wt 4-OHT, n=258 fl/fl Vehicle, n=225 fl/fl 4-OHT cells across 2 ( wt/wt ), 4 ( fl/wt ), and 3 ( fl/fl ) biological replicates. P-values derived from unpaired t-test of normal cell values between treatment groups for each genotype. c, Images of anaphases in control (untreated) or doxycycline-treated HeLa cells expressing tetracycline-inducible Cas9 (TetOn-Cas9) and single-guide RNA (sgRNA) that are Non-targeting (NT) or specific for SETD2 . Cells are counterstained with Hoechst (grey). Lagging (blue) and bridging (orange) chromosomes occur in cells lacking SETD2. Scale bars are 5μm. d, Quantification of chromosome segregation errors during anaphase and early telophase in cells described in c . n=183 sgNT control, n=190 sgNT dox., n=198 sgSETD2.1 control, n=240 sgSETD2.1 dox., n=187 sgSETD2.2 control, n=246 sgSETD2.2 dox. cells across 3 biological replicates. P-values derived from unpaired t-test of normal cell values between treatment groups for each sgRNA background. e, Image of Setd2 fl/fl MEFs treated with 4-OHT (at left), showing nuclear defects that occur in interphase after three days treatment. Scale bars are 50μm (top images) and 5μm (greyscale images at bottom). f, Quantifications of nuclear phenotypes from images described in e for Setd2 MEFs. n=450 wt/wt vehicle, n=617 wt/wt 4-OHT, n=650 fl/wt vehicle, n=509 fl/wt 4-OHT, n=736 fl/fl Vehicle, n=687 fl/fl 4-OHT cells across 3 biological replicates. P-values derived from unpaired t-test of normal cell values between treatment groups each genotype. g, Quantification of interphase bridges observed in cell images described in e . P-values derived from unpaired t-test of normal cell values between treatment groups each genotype. Error bars are standard deviation (s.d.).

Article Snippet: HeLa TetOn-Cas9 cells were transduced with 8mg/mL polybrene (Millipore Sigma, TR-1003) and viral supernatant for guides targeting SETD2 or non-targeting control sgRNA (Addgene, 80189).

Techniques: Control, Derivative Assay, Expressing, Standard Deviation