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. 2004 Aug;15(8):3642–3657. doi: 10.1091/mbc.E03-11-0796

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Copyright © 2004, The American Society for Cell Biology

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Figure 1. — Silencing of pericentrin A and B causes mitotic defects. (A) SAOS cells with reduced pericentrin after siRNA treatment stained with a pericentrin antibody (M8, green), which recognizes both isoforms of pericentrin (Pc A/B) and DNA (DAPI, blue). (A′) Same field as in A costained for microtubules (α tubulin, red). (B) Cells with reduced lamin A stained for lamin A (green) and DNA. (B′) Same field as in B stained for centrosomes with 5051 autoimmune sera (green) and microtubules (red). (C and C′) Cells with reduced pericentrin B (targeting the C-terminal PACT domain) stained with pericentrin B-specific antibody, Pc B, (C, green), together with microtubule label (C′) or pericentrin antibody that recognizes both isoforms (C″). (A, B, C, and C″) Maximum projection of z series without deconvolution. (A′, B′, and C′) Maximum projection of deconvolved z series. Note the improvement in the resolution of the DNA. Arrows indicate cells expressing near normal levels of the targeted protein. (D) Western blots of crude cell lysates demonstrating reduction of pericentrin B (Pc B) but not pericentrin A (Pc) by using Pc B-specific siRNA or reduction of both isoforms relative to lamin A siRNA control, by siRNA targeting Pc A and Pc B (Pc A/B). Pericentrin isoforms probed with Pc A/B anti-pericentrin antibody (M8). α Tubulin loading control probed with DM1α anti-alpha tubulin antibody. (E) Graph showing percentage of mitotic SAOS cells with spindle defects (monopolar, multipolar, and reduced astral microtubules) at 48 and 72 h after siRNA treatment. One hundred to 150 mitotic cells scored per bar. (F) Graph indicating percentage of mitotic cells with monopolar spindles at 48 and 72 h. (G) Cells with reduced pericentrin A/B (Pc A/B, red) retain the centriole marker GT335 (green) in interphase and mitosis. (H) γ Tubulin in cells with reduced pericentrin A/B seems largely unchanged at centrosomes in interphase but is reduced at spindle poles. Pc A/B (red), γ tubulin (GTU88, green), and DAPI (blue). (G–H) Maximum projection of z series with no neighbor deconvolution. Arrows indicate cells with near normal staining levels of pericentrin. Asterisks indicate mitotic cells. Mitotic cells are show at higher magnification in insets.

Figure 1. — Silencing of pericentrin A and B causes mitotic defects. (A) SAOS cells with reduced pericentrin after siRNA treatment stained with a pericentrin antibody (M8, green), which recognizes both isoforms of pericentrin (Pc A/B) and DNA (DAPI, blue). (A′) Same field as in A costained for microtubules (α tubulin, red). (B) Cells with reduced lamin A stained for lamin A (green) and DNA. (B′) Same field as in B stained for centrosomes with 5051 autoimmune sera (green) and microtubules (red). (C and C′) Cells with reduced pericentrin B (targeting the C-terminal PACT domain) stained with pericentrin B-specific antibody, Pc B, (C, green), together with microtubule label (C′) or pericentrin antibody that recognizes both isoforms (C″). (A, B, C, and C″) Maximum projection of z series without deconvolution. (A′, B′, and C′) Maximum projection of deconvolved z series. Note the improvement in the resolution of the DNA. Arrows indicate cells expressing near normal levels of the targeted protein. (D) Western blots of crude cell lysates demonstrating reduction of pericentrin B (Pc B) but not pericentrin A (Pc) by using Pc B-specific siRNA or reduction of both isoforms relative to lamin A siRNA control, by siRNA targeting Pc A and Pc B (Pc A/B). Pericentrin isoforms probed with Pc A/B anti-pericentrin antibody (M8). α Tubulin loading control probed with DM1α anti-alpha tubulin antibody. (E) Graph showing percentage of mitotic SAOS cells with spindle defects (monopolar, multipolar, and reduced astral microtubules) at 48 and 72 h after siRNA treatment. One hundred to 150 mitotic cells scored per bar. (F) Graph indicating percentage of mitotic cells with monopolar spindles at 48 and 72 h. (G) Cells with reduced pericentrin A/B (Pc A/B, red) retain the centriole marker GT335 (green) in interphase and mitosis. (H) γ Tubulin in cells with reduced pericentrin A/B seems largely unchanged at centrosomes in interphase but is reduced at spindle poles. Pc A/B (red), γ tubulin (GTU88, green), and DAPI (blue). (G–H) Maximum projection of z series with no neighbor deconvolution. Arrows indicate cells with near normal staining levels of pericentrin. Asterisks indicate mitotic cells. Mitotic cells are show at higher magnification in insets.