1. Volume 7, Issue 4, Pages 715-727 (April 2001)
The Large Subunit of Replication Factor C Promotes Cell Survival after DNA Damage in an LxCxE Motif– and Rb-Dependent Manner 
V Pennaneach, I Salles-Passador, A Munshi, H Brickner, K Regazzoni, F Dick, N Dyson, T.-T Chen, J.Y.J Wang, R Fotedar, A Fotedar 
Molecular Cell 
Volume 7, Issue 4, Pages (April 2001)
DOI: /S (01)

2. Figure 1
RF-Cp145 Promotes Cell Survival after DNA Damage in an LxCxE-Dependent Manner
(A) RF-Cp145 promotes cell survival after UV irradiation. MCF-7 cells were transiently transfected with eukaryotic expression vector encoding HA-RF-Cp145. To allow determination of cell survival, cells were cotransfected with a β-galactosidase expression plasmid. Cells were UV-irradiated 36 hr posttransfection, and the percentage of surviving cells (blue cells) was determined 17 hr after irradiation by staining the cells for β-galactosidase activity.
% surviving cells = blue cells after Irradiation / blue cells after Mock treatment × 100.
In all subsequent experiments, cell survival was assayed using an identical protocol. The percentage of surviving cells from three independent experiments after UV treatment is shown. Cell survival after transfection with HA-Bcl2 (an anti-apoptotic protein) and UV irradiation is shown as a control.
(B) A representative view of cells stained for β-galactosidase. Cells were cotransfected with RF-Cp145 or control vector and either mock-treated or UV-irradiated. After UV irradiation untransfected cells die in both cultures, but the percentage surviving cells (β-galactosidase-positive cells) is higher in RF-Cp145 transfected cells as compared to control vector transfected cells.
(C) Protein expression of RF-Cp145 and Bcl2 in (A) was confirmed by immunoblotting extracts from transfected cells with HA specific antibody.
(D) RF-Cp145 promotes cell survival after UV irradiation in an LxCxE motif–dependent manner. MCF7 cells were cotransfected with β-galactosidase vector and either control vector, or vectors expressing full-length RF-Cp145, or RF-Cp145 domain A (lacks LxCxE motif), or RF-Cp145 domain B (contains LxCxE), or full-length RF-Cp145 with a mutant LxCxE motif, or RF-Cp145 domain B with a mutant LxCxE motif. The results shown are from 4 experiments. The RF-Cp145 vectors with mutant LxCxE are referred to as LxGxK in this and all other figures.
(E) RF-Cp145 inhibits UV-induced pycnotic chromatin in a LxCxE motif–dependent manner. MCF-7 cells were cotransfected with RF-Cp145 expression vectors and GFP plasmid. Pycnotic chromatin in GFP-positive cells was scored 12 hr after UV irradiation (25 J/m2). RF-Cp145 but not RF-Cp145 with a mutant LxCxE motif inhibits UV-induced pycnotic chromatin condensation. The percentage of GFP-positive unirradiated cells with pycnotic chromatin was 3.2% ± 0.3%. The results shown are from three experiments.
(F) Protein expression by various HA-tagged vectors used in (D) was determined by immunoblotting lysates from transfected cells with HA-specific antibodies
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 RF-Cp145 Binds Retinoblastoma Protein
(A) Conservation of a LxCxE motif (residues 663–667) in the large subunit of RF-C in man, mouse, and rat.
(B) Coimmunoprecipitation of endogenous Rb and RF-Cp145 from MCF-7 cell extracts. Cell extracts immunoprecipitated with Rb (left panel) and RF-Cp145 (right panel) were analyzed by cross blotting with RF-Cp145 and Rb-specific antibodies as shown. Immunoprecipitation with a preimmune rabbit antibody (control Ab) is shown. The input shown is 5 percent of the amount used for immunoprecipitation. The hyperphosphorylated forms of Rb in the input lane are identified by an asterisk.
(C) Transfected Rb and RF-Cp145 form a complex. Rb-deficient C33A cells were transfected with either Rb or with both Rb and HA-tagged RF-Cp145 vectors. Cell extracts immunoprecipitated with HA-specific antibodies and immunoblotted with Rb-specific antibodies are shown. The input shown is 5 percent of the amount used for immunoprecipitation. The hyperphosphorylated forms of Rb in the input lane are identified by an asterisk.
(D) A subdomain of RF-Cp145 (domain B) which contains a LxCxE motif binds Rb. A schematic display of proteins generated by RFC-p145 vectors is shown (left panel). The LxCxE motif maps to domain B in RF-Cp145. The ability of Rb from cell extracts to bind control GST or GST fusion proteins of RF-Cp145 domain A, or RF-Cp145 domain B, or both domains A and B of RF-Cp145 is shown (right panel). Immobilized GST fusion proteins were incubated with cell extracts. The bound proteins were eluted and immunoblotted using anti-Rb antibodies. Input shown is equivalent to the amount used for binding assay.
(E) Direct interaction between Rb and RF-Cp145 is significantly reduced in the presence of Cys706Phe Rb mutant. The Rb deletion proteins are schematically displayed (left panel). Note not all Rb mutants were used in this experiment. Immobilized purified recombinant GST-Rb which contains AB and C pockets (referred to as GST-AE), or GST-AE (Cys706Phe mutant), which does not bind LxCxE proteins, or GST were incubated with either His-RF-Cp145 domain B or with SV40 T antigen. The bound domain B (right upper panel) or SV40 T antigen (right lower panel) was analyzed by immunoblotting with specific antibodies.
(F) RF-Cp145 domain B binds to GST-Rb in an LxCxE-dependent manner. Lysates from cells transfected with HA-RF-Cp145 domain B or RF-Cp145 domain B with mutated LxCxE motif (LxGxK) were incubated with immobilized GST-Rb recombinant proteins or control GST protein. The immobilized proteins were then eluted and analyzed by immunoblotting with HA-specific antibody. The GST-Rb recombinant proteins used in this experiment included Rb containing the AB and C pockets (GST-AE), the AB pocket (GST-AB), an AE mutant with a Cys706Phe point mutation, and the C pocket (GST-SE) (listed in [E] containing residues indicated in parentheses). RF-Cp145 domain B binds to AB pocket of Rb and hence to GST-Rb AE and AB but not to SE, which only contains the C pocket. In comparison to wild-type domain B, the ability of domain B LxCxE mutant to bind to AE and to AB is dramatically reduced. Input shown is equivalent to the amount used for binding assay
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
RF-Cp145 Promotes Cell Survival after DNA Damage in an Rb-Dependent Manner
(A) The effect of exogenous Rb on the ability of RF-Cp145 to promote cell survival in Rb-deficient SAOS-2 cells after DNA damage was determined by cotransfecting RF-Cp145 (100 ng) with Rb (100 ng) expression plasmids and 50 ng of β-gal plasmid. The percentage of surviving cells after UV treatment (25 J/m2) is shown. RF-Cp145 but not RF-Cp145 with mutated LxCxE motif promotes cell survival in SAOS-2 cells only in the presence of cotransfected Rb. Note that more surviving cells are observed upon cotransfection with Rb and control vector in comparison with control vector alone (compare control vector −Rb and +Rb). The results shown are from 3 independent experiments.
(B) Expression of wild-type RF-Cp145, RF-Cp145 with a mutant LxCxE in the presence or absence of cotransfected Rb was determined by immunoblotting cell lysates with HA-specific antibodies.
(C) The efficacy of RF-Cp145 to promote cell survival in SAOS-2 cells after DNA damage is dependent on the amount of coexpressed Rb. SAOS-2 cells were cotransfected with either 100 ng of control vector, or vector expressing RF-Cp145, or RF-Cp145 with a mutant LxCxE motif in the presence of varying amounts of Rb expression vector (50 ng, 75 ng, and 100 ng). Similar results were obtained in two other experiments.
(D) RF-Cp145 inhibits UV (25 J/m2) induced pycnotic chromatin in an Rb-dependent manner. SAOS-2 cells were transfected with RF-Cp145, or RF-Cp145 with a mutant LxCxE motif or control vector in the presence of or absence of cotransfected Rb. All cells were transfected with GFP plasmid. RF-Cp145 inhibited UV-induced pycnotic chromatin in SAOS-2 cells only if the cells were cotransfected with Rb. The results from three independent experiments are shown. The percentage of GFP-positive unirradiated cells with pycnotic chromatin was <2%.
(E) Mapping the regions in RF-Cp145 required to promote cell survival after UV irradiation (25 J/m2) in an Rb-dependent manner in Rb deficient C33A cells. C33A cells were co-transfected with RF-Cp145 (500 ng) and β-galactosidase (200 ng) vectors in the presence or absence of Rb (500 ng). The panel of RF-Cp145 vectors tested include full length RF-Cp145 (1-1148), the LxCxE motif containing domain B ( ), the domain required for interaction of RF-Cp145 with other RF-C subunits ( ) and a C-terminal deletion mutant of RF-Cp145 (1-728) which lacks the RF-C interaction domain. The results shown are from 3 independent experiments
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Rb Mutants which Cannot Bind LxCxE Proteins Fail to Promote Cell Survival after UV Irradiation
(A) SAOS-2 cells were co-transfected with 50 ng β-gal plasmid and with varying amounts (50, 75, 100, 150 or 200 ng) of pCMV-Rb wild type or pCMV Rb N757F mutant or a 200 ng control empty vector. The percentage of β-galactosidase positive surviving cells 17 hrs after UV treatment (25 J/m2) from 3 independent experiments are shown.
(B) SAOS-2 cells were transfected with varying amounts (50, 75 or 100 ng) pFAD-Rb wild type, or pFAD-Ile753Ala, Asn757Ala, Met761Ala Rb triple mutant (referred to as Rb Trp mutant in the figure) or 200 ng control empty vector. Note the vector backbone of the two sets of wild type and mutant Rb used in [A] and [B] are different. The percentage of surviving cells after UV treatment from three experiments are shown.
(C) The Rb N757F mutant retains the ability to inhibit cell cycle progression upon transfection into SAOS-2 cells. SAOS-2 cells were transiently transfected with pCMV-Rb wild type or pCMV Rb N757F mutant. The percentage of GFP positive cells which are also BrdU positive was used to estimate the % transfected cells in S phase. The percentage cells which are GFP negative and are BrdU positive was used to estimate the % untransfected cells in S phase.
(D) Protein expression by various Rb vectors used in [A] and [B] was determined by immunoblotting lysates from transfected cells with Rb specific antibodies
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Rb Mutant which Cannot Bind LxCxE Proteins Fails to Reconstitute the Ability of RF-Cp145 to Promote Cell Survival after Various DNA Damage Stimuli in Rb Null Cells
(A) The effect of exogenous wild type Rb or N757F Rb mutant on the ability of RF-Cp145 to promote cell survival in SAOS-2 cells after DNA damage was determined by co-transfecting RF-Cp145 (100 ng) with varying amounts of Rb expression plasmids (50 ng, 75 ng and 100 ng). The percentage of surviving cells (β-gal positive) after UV treatment is shown. RF-Cp145 can promote cell survival in SAOS-2 cells only in the presence of co-transfected wild type Rb but not N757F Rb mutant. The results shown are from 3 independent experiments.
(B) Protein expression by various vectors used in [A] was determined by immunoblotting lysates from transfected cells with HA specific antibody to detect RF-Cp145 or with Rb specific antibody.
(C) Rb reconstitutes the ability of RF-Cp145 to promote cell survival of Rb deficient C33A cells after UV irradiation (left panel), cisplatin (middle panel) and ionizing radiation (right panel). C33A cells were co-transfected with β-galactosidase plasmid (200 ng), RF-Cp145 (500 ng) and varying amounts of wild type Rb or Rb N757F mutant (0 ng, 125 ng, 250 ng and 500 ng). Cells were UV irradiated (25 J/m2), treated with 100 μM cisplatin for 17 hours of γ irradiated (60Gy). The results shown are from 3 independent experiments
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
HDAC1 and BRG1 Do Not Promote Cell Survival after DNA Damage in Rb−/− Cells
(A) The ability of RF-Cp145 to promote cell survival after UV irradiation was compared with other LxCxE motif containing cellular proteins, HDAC1 and BRG1. C33A cells were transfected with 500 ng of either control vector, or RF-Cp145 or HDAC1 or BRG1 expression vector in the presence of increasing amounts of Rb expression plasmid (0 ng, 125 ng, 250 ng or 500 ng of Rb). All cells were transfected with β-galactosidase plasmid (200 ng). Only, RF-Cp145 dramatically increased the cell survival activity of Rb. Results from three experiments are shown.
(B) Protein expression by various vectors used in [A] was determined by immunoblotting lysates from transfected cells with HA specific antibody to detect BRG1, HDAC1 and RF-Cp145 or with Rb specific antibody.
(C) Transfected Rb forms a complex with RF-Cp145 and HDAC1. C33A cells were co-transfected with Rb and either control vector, HA-RF-Cp145 (left panel), or HA-HDAC1 (right panel). Cell extracts immunoprecipitated with Rb specific antibodies were analyzed by immunoblotting with HA specific antibodies to detect associated HDAC1 or RF-Cp145 proteins. The input shown is 5 percent of the amount used for immunoprecipitation
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
The LxCxE Binding Site in Rb Is Required for DNA Damage Induced Cell Cycle Arrest
(A) UV induces cell cycle arrest in Rb deficient C33A cells in the presence of wild type Rb but not the N757F Rb mutant. C33A cells were transfected with either control vector (1 μg) or varying amounts (125, 250, 500 and 1000 ng) of wild type Rb or the N757F Rb mutant. Cells were co-transfected with puroBABE vector (100 ng) and selected with puromycin. 95% of cells transfected with Rb (wild type or mutant) after selection express Rb by immunofluorescence microscopy and <1% are Rb+ when cells are transfected with a control vector. Cells were UV irradiated (25 J/m2) and 24 hours later pulsed with BrdU for 8 hours. The % BrdU positive cells in the population was determined by immunofluorescence analysis. Only wild type Rb inhibits cell cycle progression after DNA damage. Note that Rb does not inhibit BrdU incorporation in C33A cells in the absence of DNA damage. The data shown is from four independent experiments.
(B) Protein expression was determined by immunoblotting lysates from transfected cells with Rb specific antibody
Molecular Cell 2001 7, DOI: ( /S (01) )