Journal: Oncotarget

Article Title: Basal level of FANCD2 monoubiquitination is required for the maintenance of a sufficient number of licensed-replication origins to fire at a normal rate.

doi: 10.18632/oncotarget.1796

Figure Lengend Snippet: Figure 1: Monoubiquitinated FANCD2 strongly interacts with replication origins in vivo and in vitro. Flag-wtFANCD2, but not Flag-mtFANCD2, strongly interacts with B48 and Myc2 replication origins in normally growing HEK293 cells. HEK293 cells were transiently transfected with empty vector, Flag-wtFANCD2 and Flag-mtFANCD2. 48 hr post-transfection, cells were fixed with 1% paraformaldehyde for ChIP assay. ChIP PCR primers for five origins were designed upon published sequences. As yellow arrowheads pointed, Flag antibodies pulled down a low amount of origin B48 or Myc2 -containing DNA fragments from the lysates of cells transfected with Flag-mtFANCD2, as compared to the cells transfected with Flag-wtFANCD2. Plasmids used were equally transfected into cells (Supplementary Fig. 1A2-4). Mouse-IgG1 was used as IP control and did not pull down any noticeable amount of DNA fragment. 5% of the IP-lysate was used to prepare PCR template as an input control. B. WtFANCD2, but not K561R mtFANCD2, clearly interacts with the replication origins in FA derivative cells. PD20 FA cells (FANCD2-/-) were used to establish stably-transfected cells carrying wtFANCD2 or mtFANCD2/inactivated FANCD2 with a similar cell proliferation profile with an exception of 2-3% less in S-phase population upon cells carry inactivated FANCD2 (Supplementary Fig. 1C1, c2). ChIP assays on these FA derivative cells [expressing a comparable level of FANCD2 protein (Supplementary Fig. 1C3)] were performed by using FANCD2 antibodies, which can pull down more B48 and Myc2 origin-containing DNA fragments from the lysates of cells carrying wtFANCD2, as compared to cells harboring mtFANCD2 (D2 labeled in the figure means FANCD2). C. FANCD2 is insufficiently associated with B48 and Myc2 origins in PD220 FA cells (FANCA-/-). FANCD2 ChIP assays on PD20, PD20+wtFANCD2, or PD220 cells were conducted. Equal amount of FANCD2 protein levels was confirmed in PD20+wtFACD2 cells and PD220 cells (Supplementary Fig. 1C3). FANCD2 antibodies can pull down more B48 and Myc2 origin-containing DNA fragments in PD20+wtFANCD2 cells not in PD220 cells (as a result of FANCA-/-, FANCD2 cannot be monoubiquitinated). D. Myc2 and B48 origin-containing DNA fragments strongly bind to monoubiquitinated FANCD2, but not un-monoubiquitinated or mtFANCD2. Ubiquitin-conjugated or –unconjugated FANCD2 and mtFANCD2 proteins (Supplementary Fig. 1D) were incubated with 32p-labeled DNA segments containing B48, Myc2, MCM4, B23, B17 or B13. The resulting complexes were analyzed on a 6% polyacrylamide non- denature gel. As pointed by blue and green arrowheads, B48 and Myc2 strongly bind to monoubiquitinated FANCD2 among all forms of FANCD2 proteins tested, indicated by the shifted 32p-labled DNA segments. Like non-origin fragment B13 (shown), B23 and B17, as well as MCM4 appeared to be incapable of binding well to any forms of FANCD2 proteins under the assay condition here used (not shown).

Article Snippet: Antibodies against the FANCD2, FANCL, MCM4, MCM5, MCM6 and, MCM7 were obtained from NOVUS.

Techniques: In Vivo, In Vitro, Transfection, Plasmid Preparation, Control, Stable Transfection, Expressing, Labeling, Ubiquitin Proteomics, Incubation, Binding Assay

Journal: Oncotarget

Article Title: Basal level of FANCD2 monoubiquitination is required for the maintenance of a sufficient number of licensed-replication origins to fire at a normal rate.

doi: 10.18632/oncotarget.1796

Figure Lengend Snippet: Figure 3:Normally Monoubiquitinated FANCD2 Interacts with MCM3 in an S-phase specific manner and Maintains the Optimal Function of MCM2-7-Containing Protein Complex. A. FANCD2 interacts with MCM3. Left panel: Simple blue- staining (taken in gray color) of an 8-16% gradient gel shows “an extra band” in one lane and not in the other control lane. The gel was resolved with Flag-IP elutes, which were prepared from Flag-wtFANCD2 or mtFANCD2 transfected CRL-1790 cells. “The extra band” pointed by a red arrowhead was sliced for mass spectrometry (The equal transfection efficiency of CRL-1790 cells with Flag-wt or mt FANCD2 is shown in Supplementary Fig. 3A). Right panel: CRL-1790 cells were used for transient co-transfection of GFP-fused MCM3 and Flag-wtFANCD2 or Flag-mtFANCD2 K561R. Cell lysate were prepared 48 hr post-transfection. Both Flag and GFP antibodies were used to perform reverse immunoprecipitation (IP) and Western blotting (WB). As yellow arrowheads indicated, Flag-mtFANCD2, unlike Flag-wtFANCD2, is not clearly associated with GFP-MCM3 (top part) (The transfection efficiency was comparable among transfected cells, please see IP input). The sepharose 6B chromatography (gel filtration) was performed using normally growing CRL-1790 set of stable cells (Supplementary Fig. 2B1 left panel). The endogenous FANCD2 co-peaks with endogenous MCM3 in cells carrying a normal basal level of FANCD2 monoubiquitination (middle part), but not in the cells carrying a compromised basal level of FNACD2 monoubiquitination (bottom part) (Fractions #9 and #30 correspond to the size markers of 2000 kDa and 669 kDa respectively, and the whole panel of markers is shown in Supplementary Fig. 3C). B. Confirmation of the interaction between endogenous MCM3 and FANCD2. Pool fractions from #21 to #25 were used to perform reverse IP-WB between FANCD2 and MCM3, MCM2 or MCM7, which also are a representative for MCM5, MCM6 or MCM4, respectively, given three sub-complexes (MCM3&5, MCM2&4, and MCM7&4) formed among MCM family members. FANCD2 appears to interact primarily with MCM3, but not with MCM2 or MCM7, whose antibodies pulled down a low amount of FANCD2 protein as compared to MCM3 antibodies. Vice versa, FANCD2 antibodies pulled down more MCM3 proteins as compared to the amount of MCM2 or MCM7 proteins pulled down. C. The Interaction between MCM3 and FANCD2 proteins is S-phase Specific. The reverse IP-WB of MCM3 and FANCD2 was performed using the same batch of synchronized cells used (Fig. 2A). The FANCD2 antibodies can pull down more MCM3 proteins from the lysates of the cell pool enriched in S but not in G1 phase of normal cell cycle. Similarly, MCM3 antibodies can pull down more FANCD2 proteins from the lysates of the cell pool enriched in S-phase, but not in G1 phase. D. The Size of MCM2-7 protein complex is compromised in cells carrying a low basal level of FANCD2 monoubiquitination, as compared to the one present in cells with a normal level of FANCD2 monoubiquitination. Gel-filtration was done by using nuclear extracts prepared from normally growing CRL-1790 set of stable cells carrying a normal or reduced basal level of FANCD2 monoubiquitination. The endogenous FANCD2 (marked with a green frame) and all 6 members of MCM complex (a red frame) co-peak at fraction #23 (top panel), but they do not co-peak at the same fraction, but 6 members of MCM family remain co-peak together at a different fraction (#27) (bottom panel) (Fractions #9 and #30 correspond to the size markers of 2000 kDa and 669 kDa respectively).

Article Snippet: Antibodies against the FANCD2, FANCL, MCM4, MCM5, MCM6 and, MCM7 were obtained from NOVUS.

Techniques: Staining, Control, Transfection, Mass Spectrometry, Cotransfection, Immunoprecipitation, Western Blot, Chromatography, Filtration

Journal: eLife

Article Title: Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells

doi: 10.7554/eLife.83870

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-MCM5 (rabbit polyclonal) , GeneTex , GTX33310 , WB (1:5000).

Techniques: Retroviral, Recombinant, Drug discovery, Plasmid Preparation, Expressing, Sequencing, CRISPR, Lactate Dehydrogenase Assay, Cloning, Protease Inhibitor, Software