Journal: bioRxiv

Article Title: MYCN Amplification and ATRX Mutations are Incompatible in Neuroblastoma

doi: 10.1101/379636

Figure Lengend Snippet: A ) Immunoblot of ATRX, DAXX, MYCN and β-actin across 12 cell lines. Boxes indicate ATRX mutant lines. B ) Immunoblot for ATRX and β-actin in 293T cells transfected with sc shRNA or ATRX-specific shRNAs (#9, #20). The fraction of ATRX remaining relative to sc shRNA is indicated on each lane. C ) Bar plot of the number quantity of colonies per well after transfection of each shRNA as normalized relative fold to the sc shRNA (dashed line). Each bar is the mean and standard deviation of duplicate experiments. D ) Photograph of cresyl violet–stained colonies of NB5 and U2OS cells after transfection with the shRNAs and bar plots of the number of colonies per well for duplicate experiments (mean and standard deviation). E ) Map of 2 gRNAs targeting exon 6 of ATRX . F ) Plot of the frequency of mutation for gRNA 2, with deletions in green and insertions in red, from MiSeq analysis of the PCR product spanning the target sequence of ATRX . G ) Bar plot of the proportion of MiSeq reads from the PCR product spanning the target sequence for gRNA 2 and gRNA 5 in SKNBE2 cells after 2, 10, or 14 days in culture. H ) Immunoblot for MYCN and GAPDH for the cell lines with stable integration of the doxycycline-inducible MYCN-expression construct. Numbers in parentheses above the lanes indicate the fluorescence-intensity ratio of MYCN/GAPDH. I ) Line graphs of the growth curves for each cell line in the presence (blue) or absence (black) of doxycycline. J ) Colony assay in the presence or absence of doxycycline for SKNBE2 MYCN and SKNMM MYCN cells. K ) Brightfield micrograph of SKNMM MYCN cells after 8 days in culture in the presence or absence of doxycycline. L ) Brightfield micrographs of 3 individual cells or a field of cells for SKNMM MYCN in the presence or absence of doxycycline at the indicated timepoints, relative to the starting point of the movies at 6 days after the addition of doxycycline. M ) Immunoblots of ATRX, MYCN, and β-actin in SKNMM MYCN cells without doxycycline or after 2, 4, or 45 days in culture. The escapers are pools of cells that grew in the presence of doxycycline. PCR for the MYCN transgene and a control locus (RPL0) is indicated in the lower portion of the figure. N ) Xenogen image of a mouse with an orthotopic neuroblastoma tumor, which is shown in the photograph, that arose after para-adrenal injection of a 1:1 mixture of SKNMM MYCN cells and SKNMM CONT –Luc:YFP cells. O ) Line graph of xenogen image data for individual mice after the injection described in ( N ). P ) Xenogen image of a mouse with an orthotopic neuroblastoma tumor, which is shown in the photograph, that arose after para-adrenal injection of a 1:1 mixture of SKNMM MYCN –Luc:YFP cells and SKNMM CONT cells. Q ) Line graph of xenogen image data for individual mice after the injection described in ( P ). Scale bars: K, 10 μm. Abbreviations : DOX, doxycycline; IFD, inframe deletion; sc, scrambled.

Article Snippet: To test for the NHEJ efficiency after CRISPR targeting in IMR-32 cells, we transfected the cells with the same mix of plasmids but added a control plasmid expressing gRNA targeting the hAAVS1 locus (Addgene # 41818) in a ratio of 4:4:6:1 for hAAVS1.gRNA: ATRX.gRNA: Cas-9: GFP expressing-plasmids respectively.

Techniques: Western Blot, Mutagenesis, Transfection, shRNA, Standard Deviation, Staining, Sequencing, Expressing, Construct, Fluorescence, Colony Assay, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: SETD2 safeguards the genome against isochromosome formation.

doi: 10.1073/pnas.2303752120

Figure Lengend Snippet: Fig. 1. Setd2 loss causes chromatin mis-segregation during mitosis. (A) Images of wild-type, heterozygous or homozygous deletion of Setd2 have lagging (blue arrows) and bridging (orange arrows) chromosomes during the anaphase. MEFs with no, one, or two floxed alleles of Setd2 (Wild-type/WtFlox/Wt, and Flox/Flox, respectively) were either treated with vehicle (EtOH, control in top panels) or 4-OHT (excise floxed alleles of Setd2, bottom panels) for 3 d, fixed, and counterstained with Hoechst (gray). Scale bars are 5 µm. (B) Quantification of chromosome segregation errors during the 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 an 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 nontargeting (NT) or specific for SETD2. Cells are counterstained with Hoechst (gray). Lagging (blue) and bridging (orange) chromosomes occur in cells lacking SETD2. Scale bars are 5 µm. (D) Quantification of chromosome segregation errors during the 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 three biological replicates. P values derived from an unpaired t test of normal cell values between treatment groups for each sgRNA background. (E) Image of Setd2fl/fl MEFs treated with 4-OHT (at left), showing nuclear defects that occur in interphase after 3 d treatment. Scale bars are 50 µm (top images) and 5 µm (grayscale images at the 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 three biological replicates. P values derived from an unpaired t test of normal cell values between treatment groups of each genotype. (G) Quantification of interphase bridges observed in cell images described in (E). P values derived from an unpaired t test of normal cell values between treatment groups each genotype. Error bars are SD.

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

Techniques: Control, Derivative Assay, Expressing