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. 2014 Aug 4;15(9):956–964. doi: 10.15252/embr.201438679

Figure 1. Human UBL5 is required for cell proliferation and sister chromatid cohesion maintenance.

Figure 1

A Whole-cell extracts (WCE) of HeLa cells transfected with non-targeting (CTRL) or UBL5 siRNAs for 48 h were immunoblotted with anti-UBL5 antibody.

B HeLa cells were counted at indicated times after transfection with control, UBL5, or SGO1 siRNAs [mean ± SD (error bars); N = 3].

C Sub-G1 DNA content of HeLa cells transfected with indicated siRNAs for 72 h was measured by flow cytometry analysis of propidium iodide-labeled cells [mean ± SD (error bars); N = 3].

D Mitotic indices of HeLa cells transfected with indicated siRNAs for 48 h were quantified by flow cytometry analysis of the proportion of phospho-H3(Ser10)-positive cells [mean ± SD (error bars); N = 3].

E Mitotic duration from nuclear envelope breakdown to chromatin decondensation in single live HeLa/H2B-mCherry cells transfected with control (n = 20) or UBL5 siRNA (n = 80) was monitored by time-lapse microscopy and classified as normal (45–60 min), delayed (longer than 60 min), or arrested (no decondensation).

F Time-lapse analysis of mitotic chromosome dynamics in live HeLa/H2B-mCherry cells transfected with control or UBL5 siRNA. Selected time frames of representative mitotic progression patterns and their frequencies are shown.

G Representative images of metaphase spreads from cells transfected with non-targeting (CTRL) or UBL5 siRNA. See also Supplementary Fig S1.

H Quantification of precocious sister chromatid separation in HeLa cells after knockdown of UBL5 [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted.

I HeLa cells inducibly expressing siRNA-resistant (siR) Strep-HA-UBL5 were transfected with control or UBL5 siRNA in the absence or presence of doxycycline (DOX). Two days later, cells were treated with nocodazole for 3 h and harvested for chromosome analysis.