J. Cell Sci. 128: doi: /jcs : Supplementary Material

Transcription

1 Figure S1. Microtubule and microfilament drug treatments severely interfere with mitotic spindle morphology, length and positioning. (A) GFP-fused lamin-a/c, LAP2 or BAF1 localizes to the spindle and all other cytoplasmic areas. Living HeLa cells expressing GFP-fused Lamin-A/C, LAP2 or BAF1 were viewed under a fluorescence confocal microscope and images were collected directly. Note that the spindle (arrows) localizations of these proteins. (B) Myc-tagged lamin A locates to the spindle area and cell cortex. HeLa cells expressing Myc-tagged lamin A were fixed and processed for immunofluorescence labeling with anti Myc and α-tubulin antibodies. Images were taken under a fluorescence confocal microscope. Note that the Myc-tagged lamin A was localized to the spindle (arrows) and other cytoplasmic areas with clear location on the cell cortex (arrowheads). Scale bar, 10 m. (C) HeLa cells were treated with nocodazole (Noc) for 30 minutes to depolymerize the microtubules. DMSO was used as a control. The cells were then fixed and immunostained with anti -tubulin antibody. DNA was labeled with DAPI. Note that the nocodazole treatment caused spindle depolymerized. (D) HeLa cells were treated with cytochalasin D (CD) for 30 minutes to depolymerize the microfilaments. DMSO was used as a control. The cells were then fixed and immunostained with anti - and - tubulin antibodies. DNA was labeled with DAPI. Note that the microfilament drug treatment caused abnormal spindle morphology and positioning. The spindles were rotated, eccentric and short, and the poles of many

2 spindles were unfocused. (E) HeLa cells were treated with Y for 30 minutes to inhibit myosin II activity to interfere the interaction of the microfilaments with myosin II. Note that the treatment caused the same results as in A. (F) HeLa cells were treated with Latrunculin A for 30 min to inhibit the microfilament polymerization. Note that the treatment caused the same results as in A. (G) Statistic analysis of different abnormal spindle types caused by the treatments in C, D and E. Three independent experiments were carried out and two hundred mitotic cells were counted in each sample. p<0.05. Scale bar, 10 m.

3 Figure S2. One protein knockdown of Lamin-A/C, LAP2α and BAF1 reduces the localizations of the other two to the spindle and the cell cortex. (A) Lamin-A/C, BAF1 and LAP2 knockdown efficiency analysis. HeLa cells were transfected with control sirna and sirna for BAF1, lamin A/C or LAP2α for 72 hours. The cells were then collected and the cell lysates detected for BAF1, LAP2α and lamin A/C by western blotting with specific antibodies. GAPDH serves as a loading control. (B) HeLa cells were transfected with sirna to knock down LAP2α, followed by immunostaining with anti BAF1 and lamin-a/c (LA/C). The knockdown efficiency was checked by LAP2α staining with a specific antibody. Note that the spindle and cell cortex localizations of BAF1 and lamin-a/c in control cells but not in LAP2α knockdown cells. (C) HeLa cells were transfected with sirna to knock down lamin-a/c, followed by immunostaining with anti BAF1, LAP2α and α-tubulin. The knockdown efficiency was checked by lamin-a/c (LA/C) staining with a specific antibody. Note the spindle and cell cortex localizations of BAF1 and LAP2α in control cells but not in lamin-a/c (LA/C) knockdown cells. (D) HeLa cells were transfected with sirna to knock down BAF1, followed by immunostaining with anti LAP2α and lamin-a/c antibodies. The knockdown efficiency was checked by BAF1 staining with a specific antibody. Note the spindle and cell cortex localizations of LAP2α and lamin-a/c in control cells. The spindle and cell cortex localizations of LAP2α and lamin-a/c in BAF1 knockdown cell were largely reduced. DNA in all these cells was labeled with DAPI. Scale bar, 10 m. (E) The triple RNAi of lamin A/C, LAP2α and BAF1 does not change lamin B1 protein level. HeLa cells were transfected with sirna to knock down LAP2α,BAF1 and lamin A/C,and 72 hours after transfection we collected the cell lysates and detected lamin B1 protein level through Western blot staining with an lamin B1 specific antibody. The knockdown efficiency was checked by LAP2α,BAF1 and lamin A/C Western blot staining with specific antibodies.

4 Figure S3. Lamin-A/C, BAF1 and LAP2 knockdown caused abnormal spindle formation and mis-positioning. (A) Montage of a normal cell cycle cells expressing both GFP- -tubulin and RFP-H 2 B. The cells were viewed as a normal control under a fluorescence microscope and images were captured every three minutes. Note that the transition of the cell cycle from metaphase to the end of telophase lasted about 33 minutes. (B) BAF1 protein knockdown caused abnormal spindle formation and mis-positioning. HeLa cells expressing both GFP- -tubulin and RFP-H 2 B were transfected with sirna for BAF1 knockdown. The images were captured every three minutes. Note that the spindle was short, eccentric and rotated, and that the metaphase-anaphase transition did not carry on. (C) LAP2 protein knockdown caused abnormal spindle formation and mis-positioning. HeLa cells expressing both

5 GFP- -tubulin and RFP-H2B were transfected with sirna for LAP2 knockdown. The images were captured every three minutes. Note that the spindle was short, eccentric and rotated, and that the metaphase-anaphase transition did not carry on. (D) Lamin-A/C protein knockdown caused abnormal spindle formation and mis-positioning. HeLa cells expressing both GFP- -tubulin and RFP-H2B were transfected with sirna for lamin-a/c knockdown. The images were captured every three minutes. Note that the spindle was short, eccentric and rotated, and that the metaphase-anaphase transition did not carry on. Scale bar, 10 m. (E and F) The angle = arctan (z/x) were used to measure the spindle pole elevation angle in X-Z axes. (E) α-tubulin (α-tub) was immunostained with specific antibodies. DNA was labeled with DAPI. Different layers of the mitotic spindle along Z-axis in lamin-a/c, LAP2α and BAF1 triple knockdown cells were shown. Note that the two spindle poles in triple RNAi cell were not in the same layer parallel with the coverslip surface. (F) Three dimensional reconstruction shown the elevation angle α = arctan (z/x).

6 Figure S4. RNAi triple knockdown of lamin-a/c, BAF1 and LAP2 knockdown caused the connection failure of the astral microtubules with the cell cortex in prometaphase and metaphase cells. (A) Prometaphase control of irrelevant RNAi. HeLa cells grown in glass bottom dishes were co-transfected with irrelevant sirna and mcherry-h2b. 72h after the transfection, the vector encoding the microtubule positive end binding protein EB3 tagged with GFP was introduced for 24 hours, followed by time-lapse confocal microscopy. The merged images of distinct layers in Z-axis at each of the different time points are shown. Note that the EB3-labeled astral microtubules of the prometaphase cells could continuously reach the cell cortex (Arrows). (B) RNAi triple knockdown of lamin-a/c, BAF1 and LAP2 knockdown in prometaphase cells. HeLa cells grown in glass bottom dishes were co-transfected with triple sirnas for lamin-a/c, LAP2α, BAF1 and RFP-H2B for 72 hours. GFP-EB3 was then introduced into the cells for 24 hours, followed by time-lapse microscopy. Note that in live cells the astral microtubules could not efficiently link to the cell cortex in lamin-a/c, LAP2α and BAF1 triple knockdown

7 cells (arrowheads). (C) Metaphase control of irrelevant RNAi. HeLa cells grown in glass bottom dishes were co-transfected with irrelevant sirna and mchery-h2b. 72h after the transfection, the vector encoding the microtubule positive end binding protein EB3 tagged with GFP was introduced for 24 hours, followed by time-lapse microscopy. Note that the EB3-labeled astral microtubules of the metaphase cells also could continuously reach the cell cortex (Arrows). (D) RNAi triple knockdown of lamin-a/c, BAF1 and LAP2 in metaphase cells. HeLa cells grown in glass bottom dishes were co-transfected with triple sirnas for lamin-a/c, LAP2α, BAF1 and RFP-H2B for 72 hours. GFP-EB3 was then introduced into the cells for 24 hours, followed by time-lapse microscopy. Note that in live cells the astral microtubules could not efficiently link to the metaphase cell cortex in lamin-a/c, LAP2α and BAF1 triple knockdown cells (arrowheads) and that the spindle is obviously eccentric the cell center. (E-G) Isolation of mitotic cells by nocodazole treatment and shaken-off of the cell culture. (E) HeLa cells were double treated with thymidine, released for 10 hours and supplemented with 100 nm/ml nocodazole for another 6 hours. The round-up mitotic cells were shaken-off and fixed and stained by DAPI. (F) Representative high magnification image of the mitotic cells in (E). (G) The cell lysates from asynchronized interphase and nocodazole-treated mitotic cells were probed with an antibody against phosphorylated histone H3 Ser10 (H3pS10), and GAPDH was probed as a loading control. Note that only the lysate of the nocodazole-treated mitotic cells was positive of phosphorylated histone H3 Ser10 staining. Scale bar, 10 m.