Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

Genes with altered expression in G0S2 null MEFs as compared to wild-type MEFs

PDF file, 90K, Repression of UBP43 mRNA levels reduces cyclin D1 protein, but not mRNA expression.

Supplemental File S1. The siRNA sequences and real-time qPCR primer sequences.

PDF file, 76K, Reduction of UBP43 induces apoptosis and decreases cyclin D1 levels in ED-1L lung cancer cells.

The siRNA sequences used in this study.

Expression of G0S2 in breast cancer versus normal breast tissue

<p>Supplemental Fig. S3. (A) Schematic diagram showing the relationship between CDK2, CP110, activated KRAS and anaphase catastrophe. (B) Engineered overexpression of CP110 was detected in A549, Hop62, H522 and H460 cells (versus control transfectants) using anti-HA antibody after 24 and 48 hours of transfection. A representative immunoblot for each cell line is displayed.</p>

Supplementary Fig. S1 from UBE1L causes lung cancer growth suppression by targeting cyclin D1

Ser 551 and Ser 906 of CP110 are not critical for protecting cells from undergoing anaphase catastrophe. (A) ED-1 cells overexpressing wild-type CP110 (CP110-WT), 8 CDK phosphorylation sites mutated CP110 (CP110-MUT), 10 CDK phosphorylation sites mutated CP110 (CP110-ALL MUT) or an empty vector were treated with the indicated dosage of seliciclib. After 24 hours treatment, ED-1 cells were fixed and scored for multipolar anaphases (NS, not significant). (B) Schematic of CP110-WT, CP110-MUT (8 sites) and CP110-ALL MUT (10 sites) sequences.

supplementary figure legend; Supplementary Fig. 1. ED1 lung cancer cells were incubated with dinaciclib; Supplementary Fig. 2. See video file; Supplementary Fig. 3. H1299 were transfected with CDK1, CDK2-expressing vector or insertless vector control; Supplementary Fig. 4. High-throughput analysis of 108 NSCLC cells to determine IC50 and IC70 values for dinaciclib.

Knock-down of Usp33 in murine lung cancer cells does not affect CP110 protein expression. Effects of Usp33 knock-down at 24,48, 72 and 96 hours on CP110 expression in ED-1 cells.

Effects of CP110 phosphorylation sites on CP110 protein stability. KRAS wild-type ED-1 cells were transfected with CP110-WT or CP110-MUT 24 hours before treatment with or without cycloheximide (CHX, 40μg/ml) for indicated time periods. Expression of CP110 in each time point was shown by immunoblot analyses and ImageJ quantifications.

<p>Supplemental Fig. S1. Effect of wild-type or a phosphorylation sites mutant CP110 species on responses to CDK2 inhibition. (A) Overexpression of CP110 was detected in ED-1 cells with an anti-HA antibody 24 and 48 hours after transfection. (B) Overexpression of wild-type CP110 significantly reduced apoptosis induced by seliciclib treatment. ED-1 cells overexpressing CP110 were treated with the indicated dosages of seliciclib for 24 hours and analyzed for apoptosis. (C and D). Overexpression of phosphorylation-site mutated CP110 did not have a significant effect on anaphase catastrophe or apoptosis induced by CDK2 inhibition. ED-1 cells overexpressing a mutant-CP110 species were treated with the indicated dosages of seliciclib for 24 hours or transfected individually with two different siRNAs targeting CDK2 for 24 hours and (C) scored for multipolar anaphase and (D) analyzed for apoptosis. (E). Seliciclib treatment did not appreciably affect CP110 basal levels in human and murine lung cancer cells. Hop62 and ED-1 were treated with various dosages of seliclcib and CP110 levels were examined after 24 and 48 hours of treatment.</p>

Supplemental File S2. A representative video of a Hop62 cell that undergoes multipolar cell division.

<p>Supplemental Fig. S2. Overexpression of wild-type CP110, but not phosphorylation-sites mutated CP110 in LKR13 murine lung cancer cells that are driven by KRAS rescued anaphase catastrophe and reduced apoptosis induced by pharmacological CDK2 inhibition after seliciclib treatment. LKR13 cells overexpressing wild-type CP110 species or an empty vector, a phosphorylation-sites mutant-CP110 species (MUT-CP110) or an empty vector (Vector) were treated with indicated dosages of seliciclib. After 24 hours treatment, LKR13 cells were (A) scored for multipolar anaphase and (B) analyzed for apoptosis, as detected by Annexin V:FITC and 7-aminoactinomycin D (7-AAD) staining.</p>

Movie video files of HOP62 cells as described in the Methods.

PDF file, 75K, Deregulation of UBP43 affects immortalized bronchial epithelial cell growth, apoptosis and cyclin D1 levels.

Abstract UBE1L is the E1-like ubiquitin-activating enzyme for the IFN-stimulated gene, 15-kDa protein (ISG15). The UBE1L-ISG15 pathway was proposed previously to target lung carcinogenesis by inhibiting cyclin D1 expression. This study extends prior work by reporting that UBE1L promotes a complex between ISG15 and cyclin D1 and inhibited cyclin D1 but not other G1 cyclins. Transfection of the UBE1L-ISG15 deconjugase, ubiquitin-specific protein 18 (UBP43), antagonized UBE1L-dependent inhibition of cyclin D1 and ISG15-cyclin D1 conjugation. A lysine-less cyclin D1 species was resistant to these effects. UBE1L transfection reduced cyclin D1 protein but not mRNA expression. Cycloheximide treatment augmented this cyclin D1 protein instability. UBE1L knockdown increased cyclin D1 protein. UBE1L was independently retrovirally transduced into human bronchial epithelial and lung cancer cells. This reduced cyclin D1 expression and clonal cell growth. Treatment with the retinoid X receptor agonist bexarotene induced UBE1L and reduced cyclin D1 immunoblot expression. A proof-of-principle bexarotene clinical trial was independently examined for UBE1L, ISG15, cyclin D1, and Ki-67 immunohistochemical expression profiles in pretreatment versus post-treatment tumor biopsies. Increased UBE1L with reduced cyclin D1 and Ki-67 expression occurred in human lung cancer when a therapeutic bexarotene intratumoral level was achieved. Thus, a mechanism for UBE1L-mediated growth suppression was found by UBE1L-ISG15 preferentially inhibiting cyclin D1. Molecular therapeutic implications are discussed. [Mol Cancer Ther 2008;7(12):3780–8]

Effects of Ser 170 and Thr 194 CDK phosphorylation sites of CP110 on CDK2-inhibitior-mediated anaphase catastrophe in human lung cancer cells. (A) Hop62 cells were transfected with CP110-MUT with indicated phosphorylation sites returned to wild-type sequence. Twenty four hours after transfection, cells were treated with seliciclib (10 μM) for 24 hours and fixed and scored for multipolar anaphases. (B) Hop62 cells were transfected with a CP110-WT vector with indicated phosphorylation sites replaced with alanine residues. Twenty four hours after transfection, cells were treated with seliciclib (10 μM) for 24 hours and fixed and scored for multipolar anaphases. The symbols indicate * P < 0.05 and ** P < 0.01.