Supplementary MaterialsS1 Fig: Karyotype analysis of cultured cell lines. different cell lines. Radial position of chromosomes in nuclei from seven different Drosophila cell types, determined by shell analysis with five shells of equivalent volume, where shell 1 is definitely closest to the nuclear periphery and 5 is the nuclear center. Data demonstrated represent one technical replicate (n300 cells). These data were confirmed by two additional technical replicates.(TIF) pgen.1007393.s003.tif (315K) GUID:?6A387827-345D-44C5-BF09-DCF4DD1A8232 S4 Fig: Condensin II depletion in Kc167 cells. (A) qPCR confirming efficient knockdown of Cap-H2, Rad21, and Barren in Kc167 cells. Relative mRNA levels were normalized to levels in control RNAi samples and then to Take action5c levels. Error bars were determined across three different biological replicates. (B) IF/Seafood in Kc167 cells depleted of Cap-H2, Barren, or Rad21. Heterochromatin is normally tagged with anti-H3K9me2 antibody (green), all chromosome Oligopaints are proven in magenta, and heterochromatin Seafood probes (Het) labeling the AATAT, AATAG, AACAC, 359, and dodeca satellites in grey. Scale club equals 5 m. n 500 cells per condition. (C) Tukey container plot displaying the mean and distribution (minus outliers) of the amount of Het foci. Data proven are from an individual natural replicate (n 500 cells each). These total outcomes had been verified by two extra natural replicates, respectively. ***p 0.0001; Mann-Whitney check. (D) Quantification of mitotic chromosome spreads performed after depletion of Dark brown (control) or Cap-H2 in Kc167 cells. 98% of control cells and 93% of Cap-H2 depleted cells demonstrated the standard Kc167 karyotype (find S1 Fig; p = 0.33; Fishers Specific Check). (E) Scatter story of nuclear quantity (X-axis) versus 2L CT quantity or X-2L overlap quantity (Y-axis) of Cap-H2 depleted cells. Chromosome 2L quantity data are proven in blue, while X-2L overlap data are proven in grey. R2 values had been computed in Prism. n = 534 cells. (F) qPCR confirming effective knockdown of Cap-H2, Cap-D3, and SMC2 in Kc167 cells. Comparative mRNA levels had been normalized to amounts in charge RNAi samples and to Action5c amounts. (G) Oligopaints labeling chromosomes X (green), 2L (crimson), and 2R (cyan) on consultant Kc167 cell nuclei depleted of PKC 412 (Midostaurin) Dark brown (control), Cap-D3, or SMC2. Dashed lines represent the nuclear edge. Scale pub equals 5 m. (H) Tukey package plot showing CT quantities after depletion of Condensin II subunits. The data demonstrated represent one biological replicate (n400 cells ICOS per RNAi). These data were confirmed by two additional biological replicates. (I) Pub graph showing common contact frequency between the X and 2L CTs after depletion of condensin II subunits. Error bars represent the standard deviation of three biological replicates (each PKC 412 (Midostaurin) n400 cells per RNAi). (J) Histogram showing X-2L CT overlap like a percent of X CT volume. Binned data from a single biological experiment are demonstrated (n 400 cells per RNAi). These results were confirmed by two additional biological replicates. ***p 0.0001; Mann-Whitney test. (K) Average 2L radial position in the nucleus determined by shell analysis with five shells of equivalent volume, where shell 1 is definitely closest to the nuclear periphery and 5 is the nuclear center. Averages from a single biological experiment are demonstrated (n 400 cells per RNAi). These results were confirmed by two additional biological replicates. ***p 0.0001; Mann-Whitney test.(TIF) pgen.1007393.s004.tif (802K) GUID:?1E6CFF3E-D019-4096-8A11-28C4842A8FE8 S5 Fig: Cap-H2 depletion in BG3 cells. (A) Oligopaints labeling chromosomes X (green), 2L (reddish), and 2R (cyan) on representative BG3 cell nuclei depleted of Brown (control), Cap-H2, or slmb. Dashed lines represent nuclear edge. Scale pub equals 5 m. n350 cells per RNAi. (B) qPCR confirming efficient knockdown of Cap-H2 and SLMB in BG3 cells. Relative mRNA levels were normalized to levels in control RNAi samples and then to Take action5c levels. (C) Tukey package plot showing CT quantities after depletion of Cap-H2 and slmb in BG3 cells. The data demonstrated represent one biological replicate (n350 cells per PKC 412 (Midostaurin) RNAi). These data were confirmed by one additional biological replicate. (D) Pub graph showing common contact frequency between the X and 2L CTs (remaining) or X and 2R CTs (right) after depletion of Cap-H2 in BG3 cells. Error bars represent the standard deviation of two biological replicates (each n350 cells per RNAi). (E) Histogram showing X-2L CT overlap like a percent of X CT volume in BG3 cells depleted of Brown (control) or Cap-H2. Binned data from a single biological experiment are demonstrated (n 350 cells per RNAi). These results were confirmed by one additional biological replicate. ***p 0.0001; Mann-Whitney test. (F) Average 2L radial.