Coleman et al., Supplementary Figure 1

Transcription

1 Coleman et al., Supplementary Figure 1 BrdU Merge G1 Early S Mid S Supplementary Figure 1. Sequential destruction of CRL4 Cdt2 targets during the G1/S transition. HCT116 cells were synchronized by sequential thymidine and nocodazole treatment and fixed at 2.5 h (G1), 4 h (early S), or 5.5 h (mid S) post nocodazole release. Cells were labeled with BrdU 3 minutes prior to fixation and staining with the indicated antibodies. Scale bar = 1 mm.

2 % Remaining Cell cycle length (h) Cell Number Coleman et al., Supplementary Figure 2 C) -CFP-HA UV (min): YFP-HA- -CFP-HA Anti -HA Anti- (light) Doxycycline (ng/ml): CFP-HA YFP-HA- Anti Anti -CFP-HA YFP-HA- Anti- (dark) Anti D) ng/ml 2 ng/ml ng/ml 2 ng/ml Time (min) YFP -CFP E) C 4C Doxycycline (ng/ml) 2 75 ng/ml ng/ml ng/ml dox dox dox Supplementary Figure 2. YFP- and -CFP fusion protein expression and validation. ( Asynchronously proliferating U2OS cells expressing -CFP-P2A-YFP- were treated with 2 J/m 2 UV and collected at the indicated times. The asterisk () marks the predicted position of the uncleaved fusion which is undetectable. ( Quantifications of immunoblots from A. (C) U2OS cells were treated with the indicated concentrations of doxycycline to induce expression of YFP- and -CFP fusion proteins; fusions were detected by immunoblotting. (D) Cells treated with the indicated doxycycline concentrations as in (C) were analyzed by flow cytometry for DNA content. (E ) Live cell movies of cells induced with the indicated doxycycline concentrations were analyzed for cell cycle length. Twenty cells ( ng/ml and 2 ng/ml) and 19 (75 ng/ml) cells were counted from one mitotic event to the next mitotic event. Error bars indicate standard deviations.

3 Cell Number HA- ln (% remaining) band intensity % remaining Coleman et al., Supplementary Figure 3 Fraction loaded (-UV) Time (min) post-uv i) 1. ii) (dark) (light) tubulin iii) fraction loaded Time (min) Time (min) post-uv y = -.41x t 1/2 () = 11 min +CHX UV (min): PR-Set7 HA CHX C) UV (J/m 2 ): HA- vector D) J/m 2 1 J/m 2 2 J/m 2 5 J/m 2 E) HeLa HCT116 UV (min): PR-Set F) K153A UV (min): HA- K153A C 4C WT-HA- (no UV) Supplementary Figure 3. protein degradation is independent of DNA damage-inducible expression. (. Example of semi-quantitative immunoblot analysis of substrate degradation kinetics. (Lanes 1-6, (i): Two-fold serial dilutions of HCT116 cell lysate were immunoblotted for endogenous and tubulin (light and dark exposures are shown). Lanes 7-12, (ii): HCT116 cells were irradiated with 2 J/m 2 UV, harvested at the indicated time points, and probed for endogenous and tubulin. Band intensities from multiple exposures were used to generate graphs of lysate loaded (i) and degradation (ii). (iii) Semi-log plot of % remaining values used for half-life determinations. ( HCT116 cells were subjected to 2 J/m 2 UV and treated with cycloheximide (1 µg/ml) immediately after irradiation (lanes 1-6) or left untreated (lanes 7-12). Cells were harvested at the indicated time points for immunoblot analysis. (C) Asynchronous HCT116 cells were treated with the indicated doses of UV and collected after 24 h for immunoblot analysis. (D) Cells treated as in (C) were analyzed by flow cytometry for DNA content. HCT116 cells stably expressing wild-type HA-tagged used in Figure 4A are included for comparison. (E) HeLa cells were subjected to a UV time-course and compared to HCT116 cells. (F) HCT116 cells expressing ectopic HA- deficient for TRIM39 binding (K153 were treated with 2 J/m 2 and harvested at the indicated time-points for immunoblot analysis of ectopic and endogenous levels.

4 % Remaining % Remaining Coleman et al., Supplementary Figure 4 PIP HA- UV (min): P21 PIPm tubulin 1 PIPm- endogenous Time (min) key a.a: () ---MEQRRVTDFFARRRPG PR-Set7 GKTQQNRKLTDFYPVRRSS GRKRRQTSMTDFYHSKRRL PIPm GRKRRQTSAAAAAHSKRRL C) Δ (15 546) -HA ΔPIP--HA (anti-h UV (min): tubulin 1 PIP endogenous Time (min) Supplementary Figure 4. Mutation of PIP degron sequences impairs CRL4 Cdt2 -mediated proteolysis. ( Cells expressing ectopic HA-tagged bearing alanine substitutions in amino acids were treated with 2 J/m 2 UV and harvested at the indicated time-points for immunoblot analysis. ( Alignment of the PIP degron sequences from human, PR-Set7, and. Basic residues are in magenta, hydrophobic residues in green, and the conserved T and D common to PIP degrons are in blue. (C) lacking the native PIP degron and tagged at the C-terminus was expressed from a doxycycline-inducible promoter in U2OS cells (2 ng/ml doxycycline), then analyzed as in A.

5 Cell Number Cell Number Coleman et al., Supplementary Figure 5 Doxycycline (ng/ml) : PIP --YFP YFP-WT- (-) YFP-WT PIP - -YFP Endo YFP-WT- PIP --YFP ng/ml ng/ml 1 ng/ml 1 ng/ml 3 ng/ml 3 ng/ml 2 ng/ml 2 ng/ml 2C 4C DNA Content 2C 4C DNA Content C) YFP-WT- PIP --YFP UV (min): PIP --YFP YFP-WT- Endo. 75 kda Anti- light Anti- dark Supplementary Figure 5. Validated YFP- and PIP--YFP fusion regulation and function. ( Asynchronous U2OS cells expressing either YFP (Venus)- or PIP--YFP fusions were induced with the indicated concentrations of doxycycline and then subjected to anti- immunoblot analysis. ( indicates a non-specific band that serves as a loading control.) ( Flow cytometry analysis of DNA content of cells expressing YFP fusion proteins shown in (. Doxycycline concentrations of each cell line used in the imaging analysis in Figure 5 are underlined. (C) Asynchronous U2OS cells expressing either YFP- or PIP--YFP proteins (induced with 2 ng/ml doxycycline) were treated with 2 J/m 2 UV and collected at the indicated times for immunoblot analysis.

6 Input GST GST-WT GST-PIPm Coleman et al., Supplementary Figure 6 PCNA GST- Ponceau S GST Supplementary Figure 6. PIPm (PIP degron mutant) fails to bind PCNA DNA. ( The indicated GST fusion proteins were purified from E.coli lysates with glutathione-sepharose beads and incubated with sonicated chromatin fractions from UV-irradiated 293T cells. Bound proteins were eluted in 2X SDS buffer and subjected to immunoblot analysis after normalizing for bound GST. ( Alignment of PIP degron sequences from human, PR-Set7, and PIPm. Positions previously shown to be important for CRL4 Cdt2 -mediated degradation are shown in red.

7 Coleman et al., Supplementary Figure 7 sirna: GL2 UV(min): PIP - PIP - Endo. GAPDH UBCH Anti- (dark) Anti- (light) sirna: GL2 UV(min): PIP - UBE2G1+G Endo. Endo. GAPDH Anti- (dark) Anti- (light) sirna: UBCH8 UBE2G1 UBE2G2 GAPDH Supplementary Figure 7. The PIP degron fused to is still a substrate for the -specific E2 (UBCH8). ( HCT116 stably expressing PIP - were depleted of UBCH8 or UBE2G1 +UBE2G2 as in Shibata et al Cells were treated with cycloheximide for 1 min before 2 J/m 2 UV irradiation, collected at the indicated times after UV treatment and then analyzed for ectopic and endogenous by immunoblotting; GAPDH serves as a loading control. ( Immunoblot analysis of sirna transfected cells to assess E2 depletion.

8 h post-hu Cell Number vector vector YFP-WT- PIP --YFP Coleman et al., Supplementary Figure 8 si: post-hu (h): PIP - Venus Venus-WT- tubulin Anti- light Anti- dark si-control si-control si-+ vector 12 h post-hu si-+ WT- si-+ PIP - 2C 4C DNA Content Supplementary Figure 8. HU-mediated replication-coupled destruction and recovery. ( Immunoblot and ( flow cytometry analysis of samples from HU arrest and release experiment in Figure 7D. Cells were treated with 1 nm sirna and 1 ng/ml doxycycline as indicated.