SUPPLEMENTAL FIGURE LEGENDS Figure S1. Knockdown of ...
SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Knockdown of hASUN by two independent siRNA sequences. For all panels, HeLa or U2OS cells were treated for 3 d with NT siRNA (control), hASUN siRNA#1, or hASUN siRNA#2. (A, B) Immunoblotting of cell lysates revealed a similar degree of knockdown of hASUN protein using hASUN siRNA#1 (A) or hASUN siRNA#2 (B). Tubulin was used as loading control. (C) Cells were fixed and immunostained for hASUN. The cytoplasmic hASUN signal present in control cells was lost in hASUNsiRNA#2 cells. (D) Cells were nocodazole-treated, fixed, and stained for PH3, DIC, and DNA. The perinuclear pool of dynein was markedly reduced in hASUN-siRNA#2 cells compared to control cells. (E) Cells were fixed and stained for tubulin and DNA. Cells containing >2 nuclei were scored as multinucleated. An increased percentage of hASUNsiRNA#2 cells were multinucleated compared to control cells. Scale bar, 20 µm.
Figure S2. hASUN knockdown does not inhibit microtubule depolymerization by nocodazole. HeLa cells were transfected with NT or hASUN siRNA, treated with nocodazole for 3 h or left untreated, fixed, and stained for Tubulin and PH3. (A-B, E) Without nocodazole treatment, polymerized microtubules were present in 100% of NTsiRNA and hASUN-siRNA PH3+ cells. (C-E) Following nocodazole treatment, microtubules were depolymerized in 100% of both NT-siRNA and hASUN-siRNA PH3+ cells. Scale bar, 20 µm.
Figure S3. hASUN is not required for the stability of dynein-dynactin components or dynein complex integrity. HeLa cells were transfected with either NT or hASUN siRNA, and lysates were prepared 3 d later. (A) Immunoblotting confirmed normal levels of dynein-dynactin components (DIC and DMN) following hASUN siRNA. Tubulin was used as loading control. (B) DIC-containing dynein complexes exhibited a normal migration pattern on sucrose density gradients following hASUN knockdown. Arrows indicate elution peaks of molecular weight standards.
Figure S4. Quantification of mitotic spindle defects and mitotic index after hASUN knockdown. (A) HeLa cells were transfected with NT or hASUN siRNA, fixed, and stained for Tubulin and DNA. Bar graph displays quantification of mitotic spindle defects (total on left, subdivided into classes on right) in hASUN-siRNA versus NT-siRNA metaphase cells without drug treatment. For each siRNA condition, at least 300 metaphase spindles were scored. (B) HeLa cells were transfected with NT or hASUN siRNA, fixed, and stained for PH3 and DNA. Bar graph displays mitotic index (number of PH3-positive cells/total number of cells scored, expressed as percentage). For each siRNA condition, at least 800 cells were scored per experiment. n = 3. Statistical analyses were performed using an unpaired Student’s t-test (A) or Fisher’s exact test (B). Asterisks, p
Figure S1. Knockdown of hASUN by two independent siRNA sequences. For all panels, HeLa or U2OS cells were treated for 3 d with NT siRNA (control), hASUN siRNA#1, or hASUN siRNA#2. (A, B) Immunoblotting of cell lysates revealed a similar degree of knockdown of hASUN protein using hASUN siRNA#1 (A) or hASUN siRNA#2 (B). Tubulin was used as loading control. (C) Cells were fixed and immunostained for hASUN. The cytoplasmic hASUN signal present in control cells was lost in hASUNsiRNA#2 cells. (D) Cells were nocodazole-treated, fixed, and stained for PH3, DIC, and DNA. The perinuclear pool of dynein was markedly reduced in hASUN-siRNA#2 cells compared to control cells. (E) Cells were fixed and stained for tubulin and DNA. Cells containing >2 nuclei were scored as multinucleated. An increased percentage of hASUNsiRNA#2 cells were multinucleated compared to control cells. Scale bar, 20 µm.
Figure S2. hASUN knockdown does not inhibit microtubule depolymerization by nocodazole. HeLa cells were transfected with NT or hASUN siRNA, treated with nocodazole for 3 h or left untreated, fixed, and stained for Tubulin and PH3. (A-B, E) Without nocodazole treatment, polymerized microtubules were present in 100% of NTsiRNA and hASUN-siRNA PH3+ cells. (C-E) Following nocodazole treatment, microtubules were depolymerized in 100% of both NT-siRNA and hASUN-siRNA PH3+ cells. Scale bar, 20 µm.
Figure S3. hASUN is not required for the stability of dynein-dynactin components or dynein complex integrity. HeLa cells were transfected with either NT or hASUN siRNA, and lysates were prepared 3 d later. (A) Immunoblotting confirmed normal levels of dynein-dynactin components (DIC and DMN) following hASUN siRNA. Tubulin was used as loading control. (B) DIC-containing dynein complexes exhibited a normal migration pattern on sucrose density gradients following hASUN knockdown. Arrows indicate elution peaks of molecular weight standards.
Figure S4. Quantification of mitotic spindle defects and mitotic index after hASUN knockdown. (A) HeLa cells were transfected with NT or hASUN siRNA, fixed, and stained for Tubulin and DNA. Bar graph displays quantification of mitotic spindle defects (total on left, subdivided into classes on right) in hASUN-siRNA versus NT-siRNA metaphase cells without drug treatment. For each siRNA condition, at least 300 metaphase spindles were scored. (B) HeLa cells were transfected with NT or hASUN siRNA, fixed, and stained for PH3 and DNA. Bar graph displays mitotic index (number of PH3-positive cells/total number of cells scored, expressed as percentage). For each siRNA condition, at least 800 cells were scored per experiment. n = 3. Statistical analyses were performed using an unpaired Student’s t-test (A) or Fisher’s exact test (B). Asterisks, p
