Journal: Cancer Cell

Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

doi: 10.1016/j.ccell.2020.04.013

Figure Lengend Snippet:

Article Snippet: Tritium thymidine (Methyl-3 H ) , PerkinElmer , Cat# NET027250UC.

Techniques: Recombinant, Lysis, Enzyme-linked Immunosorbent Assay, Luminex, Cell Isolation, Isolation, Transgenic Assay, Software

Journal: Advances in biological regulation

Article Title: Fhit and Wwox loss-associated genome instability: genome caretaker one-two punch

doi: 10.1016/j.jbior.2016.09.008

Figure Lengend Snippet: A) Immunoblot of Vinculin and TK1 in −/− and +/+ mouse kidney cells. B) Indirect immunofluorescence of γH2AX, before thymidine supplementation. C) Quantification of γH2AX-positive cells before thymidine supplementation. Bar graph indicates the means, and error bars represent the standard error. D) Neutral comet assay of mouse kidney cells before thymidine supplementation. Box plots of tail moments include data (WT, n = 285; Fhit −/−, n = 435) from 3 separate experiments. E) Representative photos of Fhit−/− and Fhit +/+ comet tails F) Neutral comet assay of mouse kidney cells 40 days post 10 μM thymidine supplementation. Box plots of tail moments include data (WT untreated, n = 344; WT with thymidine, n = 228; Fhit −/− untreated, n = 341; Fhit −/− with thymidine, n = 286) from 3 separate experiments. dT, thymidine.

Article Snippet: Proteins were separated by SDS gel electrophoresis, transferred to nitrocellulose membranes and immunoblotted with antisera against mouse Fhit ( Fong et al., 2000 ), GAPDH (Calbiochem), Vinculin (AbCam), and TK1 (Proteintech).

Techniques: Western Blot, Immunofluorescence, Neutral Comet Assay

Journal: Advances in biological regulation

Article Title: Fhit and Wwox loss-associated genome instability: genome caretaker one-two punch

doi: 10.1016/j.jbior.2016.09.008

Figure Lengend Snippet: A) Western blot analysis of TK1 expression in mouse kidney cells. In +/+ cells, TK1 expression decreases in parallel to loss of Fhit expression. In −/− cells, loss of Fhit results in transient TK1 downregulation, as TK1 expression increases at late passage. B) Assessment of DNA damage via neutral comet assay in mouse kidney cells. Fhit−/− NS3 is a nutritionally stressed cell line that has undergone cellular transformation in vitro and over-expresses TK1. Box plots of tail moments include: Fhit+/+ without thymidine, n=72; Fhit+/+ with thymidine, n=103; Fhit−/− without thymidine, n=282; Fhit−/− with thymidine, n=331; NS3 without thymidine, n=144; NS3 with thymidine, n=161. Thymidine supplementation (10μM) for 16 days and includes data from two separate experiments. No significant difference in levels of damage between mock and dT of Fhit+/+ (p=0.4095) and Fhit−/− NS3 (p=0.5729). dT, thymidine. C) RNA-seq data obtained from The Cancer Genome Atlas shows a negative correlation between Fhit expression and expression of enzymes involved in dTTP synthesis (TK1, RRM2, and TYMS). Red, up-regulated. Green, down-regulated. We conclude that during cancer progression, there is selective pressure to increase expression of these proteins needed for balanced dNTP pools and for optimal DNA replication.

Article Snippet: Proteins were separated by SDS gel electrophoresis, transferred to nitrocellulose membranes and immunoblotted with antisera against mouse Fhit ( Fong et al., 2000 ), GAPDH (Calbiochem), Vinculin (AbCam), and TK1 (Proteintech).

Techniques: Western Blot, Expressing, Neutral Comet Assay, Transformation Assay, In Vitro, RNA Sequencing

Journal: Advances in biological regulation

Article Title: Fhit and Wwox loss-associated genome instability: genome caretaker one-two punch

doi: 10.1016/j.jbior.2016.09.008

Figure Lengend Snippet: A) Structure of diadenosine triphosphate (Ap3A), the first recognized in vitro substrate for Fhit, and the 5’ 7-methyl-guanosine cap. B) In the 3‟ to 5‟ mRNA decay pathway, the exosome generates free m7GpppN dinucleotides that can be hydrolyzed by a scavenger decapping enzyme. The model hypothesizes that In the presence of Fhit, Fhit binds and hydrolyzes m7GpppN into m7GDP and m7GMP, which are cleared from the cell. In the absence of Fhit, free m7GpppN caps accumulate. Preferential binding of translation initiation factors to these free caps instead of capped TK1 mRNAs leads to deregulated translation of TK1 mRNA. As the model proposes, Fhit is thus a scavenger-decapping enzyme that eliminates residual cap structures to promote ribosomal binding and translation of cap-bearing mRNAs.

Article Snippet: Proteins were separated by SDS gel electrophoresis, transferred to nitrocellulose membranes and immunoblotted with antisera against mouse Fhit ( Fong et al., 2000 ), GAPDH (Calbiochem), Vinculin (AbCam), and TK1 (Proteintech).

Techniques: In Vitro, Binding Assay