Fig 1: CIA2B knockdown results in decreased rates of DNA synthesis, increased DNA damage, inhibition of cell cycle progression, and reduced cell proliferation. (A) Knockdown with two independent siRNAs results in approx. 70–75% reduction in CIA2B protein expression relative to α-tubulin in triplicate knockdown (KD) experiments. (B) IF for CIA2B confirms specific loss of spindle-associated, cytoplasmic, and nuclear fluorescence signal following anti-CIA2B knockdown. (C) siRNA-mediated KD of CIA2B expression in HeLa cells results in progressive and significant decreases (40% at 1 hr → 90% at 6 hrs) in S-phase DNA synthesis rates as detected by EdU incorporation. (D) Changes in positivity for nuclear γH2AX and pATM following CIA2B knockdown with two distinct siRNAs, expressed as relative change in percent positivity compared to scrambled siRNA control (γH2AX: siRNA1: 1.72 ± 0.09-fold; siRNA2: 1.23 ± 0.17-fold. pATM: siRNA1: 0.85 ± 0.01-fold; siRNA2: 1.28 ± 0.16-fold. (E) Apparent cell cycle delay at G1/S transition following CIA2B knockdown with two distinct siRNAs, expressed as percent of cells at each phase following siRNA (siRNA1, gray; siRNA2, black) as compared to scrambled control (Scr, white) on the left, and expressed as percent change in proportion of cells at each cell cycle phase on right: G1: siRNA1: + 14.8%; siRNA2: +12.0%; S: siRNA1: −15.0%; siRNA2: −13.3%; G2/M: siRNA1: −26.8%; siRNA2: −26.6%. (F) Decreased proliferation rate as determined by relative change in percent PHH3 positivity following CIA2B knockdown with two distinct siRNAs: siRNA1: 0.74 ± 0.04-fold; siRNA2: 0.64 ± 0.06-fold. (G) altered rates of apoptosis assayed by AC3 positivity following CIA2B knockdown with two distinct siRNAs expressed as relative change in percent positivity (siRNA1: 0.57 ± 0.01-fold; siRNA2: 3.51 ± 0.31-fold). **p < 0.02; *p < 0.05; ns = not significant.
Fig 2: Ccdc117 cellular localization and interaction with the MMS19 CIA complex. Immunofluorescence (IF) confocal images of M-phase (left) and interphase (G1) (right) HeLa cells comparing immunolocalization of Ccdc117 to that of MMS19 CIA complex components CIA2B, MMS19, and CIAO1 (magenta) vs. mitotic spindle (α-tubulin, green) and metaphase chromosomes and nuclei (Hoechst 3358, blue). Scale bar: 5 um.
Fig 3: siRNA knockdown of Ccdc117 and CIA2B results in altered G1/S and G2/M cyclin expression. (A) siRNA (gray and black bars) results in significantly elevated expression of nuclear Cyclin E (Ccdc117: +2.17 ± 0.23-fold (siRNA1), +4.13 ± 0.37-fold (siRNA2); CIA2B: 3.42 ± 0.44-fold (siRNA1), +2.73 ± 0.13-fold (siRNA2). (B) Cyclin B1 expression is unchanged following siRNA: (Ccdc117: +1.06 ± 0.02-fold (siRNA1), +1.14 ± 0.04-fold (siRNA2); CIA2B: +1.03 ± 0.02-fold (siRNA1), +0.83 ± 0.10-fold (siRNA2). **p < 0.02; ns: not significant.
Fig 4: Reciprocal co-IP experiments detect interaction of Ccdc117 with CIA2B, and interaction of both with MMS19, but not CIAO1 in cycling HeLa cell extracts. Input lysate and IP samples by antibody are shown vertically at tops of lanes; primary Ab used for Western are shown to right. MW are shown to left. * indicates a non-specific band detected by anti-CIAO1 antibody.
Supplier Page from OriGene Technologies for CIAO2B Human siRNA Oligo Duplex (Locus ID 51647)