siRNA targeting of BRG1 confirms changes in nuclear shape in MCF-10A cells.

<p>(A) Western blot analysis of MCF-10A cells transfected with three different siRNAs that target BRG1 or a control siRNA. U represents untransfected MCF-10A cells. S represents the control cells expressing a scrambled siRNA. 1, 2, and 3 represent the three siRNAs targeting BRG1. PI3 Kinase was measured as a loading control. (B) Untransfected MCF-10A cells, control MCF-10A cells transfected with a scrambled siRNA, and MCF-10A cells transfected with three different siRNAs targeting BRG1 were labeled with Lamin A/C. Arrows indicate grooves. Size bars 10 µm. (C) Quantification of grooved nuclei in untransfected, control, and BRG1 siRNA treated cells. Data represent at least three independent counts of at least 100 nuclei scored for grooves. Results are reported as means +/− standard deviation. *** represents p<0.005 as determined by Student’s t-test whether the BRG1 knockdown results were compared to untransfected or to control siRNA treated cells.</p

Induction of nuclear grooves after knockdown of BRG1 does not depend on intact actin filaments.

<p>(A) Matched individual confocal sections of representative fields of cells before and after treatment with Latrunculin B at 0.2 µg/ml for 30 minutes. Actin filaments were detected with Phalloidin-TRITC (red), while nuclei were counterstained with the DNA dye DRAQ5 (blue). Size bar 10 µm. (B) Quantification of nuclear grooves in control cells and cells treated with Latrunculin B. The means for three counts of at least 100 nuclei scored for grooves under each condition are presented +/− standard deviation. * represents p<0.05, *** represents p<0.005 as determined by Student’s t-test.</p

Induction of nuclear grooves after knockdown of BRG1 does not depend on intact microtubules.

<p>(A) Matched individual confocal sections of representative fields of cells before and after treatment with Colcemid at 0.05 µg/ml for 3 hours. Microtubules were detected with an α-Tubulin antibody (green), while nuclei were counterstained with the DNA dye DRAQ5 (blue). Size bar 10 µm. (B) Quantification of nuclear grooves in cells treated with Colcemid. The means for three counts of at least 100 nuclei scored for grooves under each condition are presented +/− standard deviation. * represents p<0.05, *** represents p<0.005 as determined by Student’s t-test.</p

The Ultrastructure of MCF-10A cells with BRG1 knockdown.

<p>Sections were prepared for electron microscopy in two orientations: perpendicular to the growth surface (panels A to F) and parallel to the growth surface (panels G and H). Representative images of the structures quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055628#pone-0055628-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055628#pone-0055628-g002" target="_blank">2</a> are shown. (A) Uninduced control MCF-10A BRG1i cells had regular oval profiles when viewed perpendicular to the growth plane. For all cells shown in this orientation, the basal side of the cell adjacent to the growth surface is toward the bottom of the micrograph, while the apical surface is facing up. (B) Irregular contours were induced in cells after BRG1 knockdown. (C) After BRG1 knockdown, some nuclei developed both irregular contours and grooves in the apical surface of the nuclei. The groove in this nucleus is shown at higher magnification in panel E. (D) Other nuclei had grooves on their apical sides but a more normal nuclear contour. Arrow indicates groove. The groove for this nucleus is shown at higher magnification in panel F. (E) Higher magnification view of the groove in the nucleus of panel C. Five nuclear pores are visible in the cleft of the groove. This shows that the wall of the groove is in an adjacent plane of section. (F) Higher magnification view of the groove in the nucleus of panel D. (G) Uninduced control MCF-10A BRG1i cells had regular oval profiles when viewed parallel to the growth plane. (H) After induction of BRG1 knockdown, these nuclei sometimes had deep invaginations of the nuclear lamina and covering envelope that projected deep into the nuclear interiors and sometimes terminated adjacent to nucleoli. Arrow indicates groove. Size bar panel E 0.5 µm. All other size bars are 2 µm.</p

HP1 is not concentrated in nuclear grooves induced by knockdown of BRG1 in MCF-10A cells.

<p>(A) Representative and matched individual confocal sections of Lamin B (green) and HP1 (red) stained cells grown in the presence or absence of doxycycline. Nuclei were counterstained with the DNA dye DRAQ5 (blue). Arrows indicate grooves in the Lamin B and Overlay images. Arrows indicate lack of corresponding grooves in the HP1 image. Size bar 10 µm. (B) Western blot analysis of HP1 and BRG1 levels. GAPDH levels were measured as a loading control.</p

The frequency of grooved and multi-lobed nuclei in MDA-MB-231 cells is independent of BRG1.

<p>(A, B) Multiple representative individual confocal sections of MDA-MB-231 cells with and without BRG1 knockdown are shown labeled with either Lamin A/C (A) or Lamin B (B). SCRAM represents the control cells expressing a scrambled sequence shRNA. Nuclei were counterstained with the DNA dye DRAQ5. Arrows indicate nuclear grooves. Thick arrows indicate multi-lobed nuclei. Size bars 10 µm. (C, D) Quantification of regular, grooved, and multi-lobed nuclei indicated no change in MDA-MB-231 cells with or without BRG1 knockdown as determined by staining with Lamin A/C (C) or Lamin B (D). Data represent three counts of at least 100 nuclei scored for grooves under each condition and are reported as the means +/− standard deviation. (E) Western blot analysis of BRG1 levels. GAPDH levels were monitored as a loading control.</p

BRG1 knockdown in MCF-10A cells increases the frequency of irregularly shaped nuclei.

<p>(A) Western blot measuring the extent of BRG1 and BRM knockdown. PI3 Kinase was measured as a loading control. Induction of BRG1 or BRM knockdown correlates with Doxycycline (DOX) dependent GFP expression. (B) Representative images of irregular and regular shaped nuclei in PAP stained cells. Size bar 50 µm. (C) Quantification of irregularly shaped nuclei in MCF-10A cells upon BRG1 or BRM knockdown. SCRAM represents the control cells expressing a scrambled sequence shRNA. Data represent the means of 6 counts totaling a minimum of 200 (SCRAM +/− DOX), 290 (BRG1i +/− DOX), or 325 (BRMi +/− DOX) cells scored per cell line +/− standard deviation. *** represents p<0.005 as determined by Student’s t-test.</p