Journal: bioRxiv

Article Title: Unlocking the Potential: FKK6 as a Microbial Mimicry-Based Therapy for Chronic Inflammation-Associated Colorectal Cancer in a Murine Model

doi: 10.1101/2024.07.30.605845

Figure Lengend Snippet: (A,B) Ames fluctuation test & Bacterial cytotoxicity. Assays was carried out in four different strains of Salmonella typhimurium, in the presence or absence of rat liver S9 fraction, as described in detail in the Materials and Methods section. CTR = background, 2AA = 2-aminoanthracene (10 μM), 9AA = 9-aminoacridine (10 μM), QUE = quercetin (30 μM), STR = streptozotocin (2.5 μM); Significance was calculated using the one-tailed Fisher’s exact test. (C) Micronucleus assay. CHO-K1 cells were incubated at 37 °C with FKK6 (0 μM; 8 μM; 16 μM; 32 μM), Mitomycin C (MTC; 0.3 μM) or cyclophosphamide (CP; 72 μM) in the presence (4 h) or the absence (24 h) of rat liver S9 fraction. The percent of micronucleated cells was calculated and the significance was determined using the t-test. (D) Mouse blood biochemistry. 30-days chronic toxicity in vivo experiment; FKK6 (0 mM; 0.5 mM; 1 mM) was dosed q.d./p.o. ; n = 6 mice per group. (E) Ki-67 intestinal expression - immunohistochemistry. Evaluated in n = 3 mice per group. (F) γ-H2AX (phospho Ser139) intestinal expression - immunohistochemistry. Evaluated in n = 3 mice per group. (G) Mice 30-days chronic toxicity experiment scheme

Article Snippet: The same tissue sections were stained and analyzed for the expression of γ-H2AX (phospho Ser139) (HistoWiz, Inc., New York City, NY, USA).

Techniques: One-tailed Test, Incubation, In Vivo, Expressing, Immunohistochemistry

Journal: Molecular Cancer

Article Title: MutS homologue hMSH5: role in cisplatin-induced DNA damage response

doi: 10.1186/1476-4598-11-10

Figure Lengend Snippet: Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation . ( A ) 293T/f-hMSH5 and 293T/f-hMSH5 Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. ( B ) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa , molecular weight ( Mr ) in thousands. ( C ) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5 Y742F , and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. ( D ) Immunoblotting analysis of the effectiveness of hRad51 or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa , molecular weight ( Mr ) in thousands. ( E ) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t -test).

Article Snippet: Antibodies used for performing IP and Western blot analysis included α-FLAG M2 (Sigma, St. Louis, MO), α-hMSH5 [ ], α-hMSH4 [ ], α-γ-H2AX (Upstate Laboratories, Inc., East Syracuse, NY), α-hRad51 (Calbiochem, Gibbstown, NJ), α-hMRE11 (Novus Biologicals Inc., Littleton, CO), α-c-Abl (BD Pharmingen, San Diego, CA), α-p-Tyr (Cell Signaling, Beverley, MA), α-α-tubulin (Sigma, St. Louis, MO), α-acetyl histone H3 (Upstate), α-histone H4 (Cell Signaling), and α-RNAPII (Upstate).

Techniques: Immunoprecipitation, Phospho-proteomics, Western Blot, Knockdown, Negative Control, Molecular Weight, Transfection, Control