Figure 7. Drosophila lamin C is... | Rockefeller University Press

Figure 7.

$Figure 7. Drosophila lamin C is necessary for PcG protein intranuclear localization and function. (A) Western blot of total protein extracts hybridized with indicated antibodies in cells transfected with indicated dsRNAs. β-Actin was used as loading control. Topoisomerase II and lamin Dm0 were used as controls to verify the integrity of nuclear matrix. Numbers indicate quantification of protein bands normalized to β-actin and relative to GFP. Data shown are from a single representative experiment of five repeats. The graph indicates quantifications of protein bands normalized to β-actin and relative to MB control. (B, left) Chromatin fractionation experiments of cells transfected as indicated in A. Equal amounts of each fraction were immunoblotted and hybridized with indicated antibodies. Positive controls: β-tubulin (S1, S2), histone H3 (S2, S3), and lamin Dm0 and topoisomerase II (S4). (right) Quantification of indicated proteins in S4 fraction, normalized to lamin Dm0. Data points represent the mean of at least two biological replicates. (C) ChIP analyses with antibodies against PC are presented as a percentage of input chromatin precipitated for the indicated region. Mock enrichment is <0.003% of the input. As negative control, we used the promoter region of brown (bw) that is repressed in S2 but is not under the control of PcG proteins (Paro and Zink, 1993). Data points represent the mean of five independent IP reactions on different chromatin preparations. (D) Quantification by real time-PCR of transcript levels, relative to GAPDH, of homeotic genes in cells transfected with indicated dsRNAs. Data points represent the mean of six independent experiments. (E, left) 3D reconstruction of single nuclei from S2 cells transfected with indicated dsRNAs and immunostained using PC antibodies. Bar, 10 µm. (right) Distribution of number of PcG protein foci per nucleus (top) and PcG foci volume (bottom; measured in µm3) among the cellular population. n > 75 from three independent experiments. (F) 3C experiments in cells transfected with indicated dsRNAs. Cross-linking frequencies, normalized to the GFP control, between the two homeotic promoters (AbdBγ and abdA) and BX-C PREs or between PRE and PRE are shown. Data points represent the mean of at least eight independent biological replicates. Two-tailed t test was applied for statistical analysis in A, B, C, D, and F. SEM is indicated. Mann–Whitney two-tailed test was applied for statistical analysis in E. Statistically relevant differences (α = 0.05): *, P < 0.05; **, P < 0.01; ***, P < 0.001.$

Drosophila lamin C is necessary for PcG protein intranuclear localization and function. (A) Western blot of total protein extracts hybridized with indicated antibodies in cells transfected with indicated dsRNAs. β-Actin was used as loading control. Topoisomerase II and lamin Dm0 were used as controls to verify the integrity of nuclear matrix. Numbers indicate quantification of protein bands normalized to β-actin and relative to GFP. Data shown are from a single representative experiment of five repeats. The graph indicates quantifications of protein bands normalized to β-actin and relative to MB control. (B, left) Chromatin fractionation experiments of cells transfected as indicated in A. Equal amounts of each fraction were immunoblotted and hybridized with indicated antibodies. Positive controls: β-tubulin (S1, S2), histone H3 (S2, S3), and lamin Dm0 and topoisomerase II (S4). (right) Quantification of indicated proteins in S4 fraction, normalized to lamin Dm0. Data points represent the mean of at least two biological replicates. (C) ChIP analyses with antibodies against PC are presented as a percentage of input chromatin precipitated for the indicated region. Mock enrichment is <0.003% of the input. As negative control, we used the promoter region of brown (bw) that is repressed in S2 but is not under the control of PcG proteins (Paro and Zink, 1993). Data points represent the mean of five independent IP reactions on different chromatin preparations. (D) Quantification by real time-PCR of transcript levels, relative to GAPDH, of homeotic genes in cells transfected with indicated dsRNAs. Data points represent the mean of six independent experiments. (E, left) 3D reconstruction of single nuclei from S2 cells transfected with indicated dsRNAs and immunostained using PC antibodies. Bar, 10 µm. (right) Distribution of number of PcG protein foci per nucleus (top) and PcG foci volume (bottom; measured in µm³) among the cellular population. n > 75 from three independent experiments. (F) 3C experiments in cells transfected with indicated dsRNAs. Cross-linking frequencies, normalized to the GFP control, between the two homeotic promoters (AbdBγ and abdA) and BX-C PREs or between PRE and PRE are shown. Data points represent the mean of at least eight independent biological replicates. Two-tailed t test was applied for statistical analysis in A, B, C, D, and F. SEM is indicated. Mann–Whitney two-tailed test was applied for statistical analysis in E. Statistically relevant differences (α = 0.05): *, P < 0.05; **, P < 0.01; ***, P < 0.001.