Monoubiquinated ID complex helps in the recruitment of downstream repair proteins, including FancD1/BRCA2, FancS/BRCA1, FancN/Palb2, FancR/Rad51, and FancJ/BRIP1. Serine 565 occurs downstream of monoubiquitination and inhibit FancD2 deubiquitination.(TIF) ppat.1007442.s001.tif (714K) GUID:?35617411-A29C-444C-A62F-5067E88C9327 S2 Fig: E6 expressing cells showed high Ub-FANCD2 & -FANCI both at baseline and after cisplatin/ MMC treatment. (A-B) Confirmation of HPV16 E6 and E7 expression Alverine Citrate by qRT-PCR (A) and immunoblot of p53 and pRb in HFK cells (B). Immunoblot showing FancD2/ FancI expression and monoubiquitination status in LXSN and E6 cells which (C) were either untreated or treated with 60ng/ml mitomycin C for 24 hr, and (D) were exposed to 10 mJ/cm2 UVB and incubated for indicated time points. (E) Immunoblot of transduced HFK cells harvested following different lengths of cisplatin treatment. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Asterisks (*) indicate a non-specific band.(TIF) ppat.1007442.s002.tif (1.1M) GUID:?130D8CC5-4D48-4422-8859-625352BD28BD S3 Fig: Determination of transcription and protein turnover rate of FancD2, FancI and UHRF1. (A) Relative mRNA expression of FanCD2, FancI and UHRF1 in HFK cells. (B-C) LXSN and E6 expressing cells were treated with 50ug/ml cycloheximide for the indicated times to determine protein turnover rate. Immunoblots (B) from a representative experiment are shown. (C) Intensities of protein bands were measured and normalized to those of GAPDH and were quantified relative to 0 hr from 2 independent experiments.(TIF) ppat.1007442.s003.tif (807K) GUID:?B3F4A46B-8C7E-4583-935F-7B294AB36B9C S4 Fig: ATR/p-S556 FancI, but not UHRF1 and PCNA help in increasing Ub-FancD2. (A-C) Immunoblots showing the effective knockdown of ATR, UHRF1 and PCNA. (D-F) Immunoblots showing FancD2 mono or de-ubiquitination status in the cells which were transfected with siControl Rabbit Polyclonal to RNF111 or respective siRNAs and were either untreated or treated with 1.5 uM cisplatin 24 Alverine Citrate hr. Levels of total FancD2 (Ub + Non-Ub) and total FancI normalized to vinculin, and ratios Alverine Citrate of phosphorylated FancI to total FancI are indicated beneath the corresponding lanes in S4D Fig.(TIF) ppat.1007442.s004.tif (194K) GUID:?2BFB8BC1-AC44-462C-BC8E-431D442DD3E4 S5 Fig: Rad51 colocalization with FancD2 in HFK-LXSN cells. (A) Schematic of I-SceI colocalization assay. The DR-GFP reporter cassette (integrated into U2OS genome) consists of two copies of nonfunctional GFP gene. The first copy is inactive due to the presence of a stop codon within the I-SceI cleavage site, while the second copy (iGFP) is truncated at both ends. Exogenous expression of I-SceI in U2OS cells with one integrated copy of the I-SceI recognition site produces a single persistent DSB. Recruitment of repair protein (green) to this enlarged pH2AX focus (red) can be visualized by IF. (B) HFK cells (transduced with LXSN) were treated with cisplatin (3 uM for 24 hr) and immunostained with FancD2 (red), Rad51 (green) and DAPI (blue). Representative images are shown.(TIF) ppat.1007442.s005.tif (962K) GUID:?C7779179-BF81-4FBE-A2E3-2D5679FCF47B S6 Fig: ATR/CHK signaling contributes to the delayed FancD2 de-ubiquitination in E6 expressing cells. (A-B) Cells were exposed to 10 mJ/cm2 UVB and incubated for indicated time points. (A) Cells were stained with DAPI and Alverine Citrate p-ATR antibody. (B) Cells were harvested at the indicated time points, and lysates were immunoblotted with antibodies to p-CHK1 and actin. (C-D) Cells were treated with 1.5uM cisplatin for 24 hr. After cisplatin withdrawal, cells were either grown in normal media (no drug) or treated with 10uM VE821 (ATR inhibitor) for indicated time points. Immunoblots of LXSN and E6 expressing cells. FancD2 Ub: non-Ub ratio are indicated beneath the corresponding lanes. pCHK1 (Serine 345) western blotting confirmed ATR inhibition by VE821.(TIF) ppat.1007442.s006.tif (1.4M) GUID:?D2B14DDF-85A6-4675-B851-2E7286725C0F S7 Fig: P53 knockdown does not change total and monoubiquitinated levels of FancD2. (A) Immunoblot showing p53 knockdown in or p53 shRNA cells compared to LXSN control. (B) Immunoblot showing FancD2 expression and monoubiquitination status in HFK LXSN and p53 knockdown cells which were either untreated or treated with 1.5 uM cisplatin for 24 hr.(TIF) ppat.1007442.s007.tif (152K) GUID:?E1561604-F540-419D-9CA7-062847A87437 S8 Fig: Delayed FancD2 deubiquitination in E6 cells is dependent on p53 degradation. (A) Immunoblots showing FancD2 mono- and deubiquitination status in HFKs which were treated with 1.5 uM cisplatin (upper panel) or 0.75uM cisplatin (lower panel) for 24 hr and allowed to repair, following cisplatin withdrawal. Initial experiments treating mutE6 cells with 1.5uM cisplatin and subsequent withdrawal did not give a clear idea (the deubiquitination pattern was more likely in between LXSN and E6). Therefore, less concentrated cisplatin (0.75uM) was used (Fig 7E)..