Journal: Science Advances
Article Title: Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis
doi: 10.1126/sciadv.ado7720
Figure Lengend Snippet: ( A ) HT29 Luc2 control (Control; Luc shRNA); HDAC5 knockdown (HDAC5kd; HDAC5 shRNA); PARP1 knockdown (PARP1kd; PARP1 shRNA); PARP1 knockdown cells (PARP1kd; PARP1 shRNA) expressing PARP1 K498R (PARP1kd/PARP1 K498R ), PARP1 K521R (PARP1kd/PARP1 K521R ), or PARP1 K498R,521R (PARP1kd/PARP1 K498R,521R ); and HDAC5 knockdown cells expressing HDAC5 S259/498A (HDAC5kd/HDAC5 S259/498A ) were orthotopically injected into the cecum wall of nude mice. After 1 week, mice were administered 5FU (10 mg/kg) every alternate day. Olaparib was also coadministered at a dose of 10 mg/kg daily as indicated. Bioluminescence imaging was performed. ( B ) Bioluminescence quantification [(A) above]. The data are representative of three independent experiments using five individual mice per group. Error bars are means ± SD from five individual mice ( n = 5 mice per group). Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). *** P < 0.001. ( C and D ) At the end of 5 weeks, the liver and lung from indicated mice [(A) above] were analyzed by ex vivo imaging (C) and hematoxylin and eosin staining (D). ( E ) At the end of 5 weeks, the blood from mice in (A) above was used to isolate genomic DNA for examining circulating tumor cells. The data are representative of three independent experiments using five individual mice per group. Error bars are means ± SD from five individual mice ( n = 5 mice per group) * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) At the end of 5 weeks, lysates of primary orthotopic tumors from (A) above were analyzed by immunoblotting for the indicated proteins. ( G and H ) Representative image of immunostaining of the indicated proteins in different grades of colon adenocarcinoma and matched normal adjacent tissue (NAT) (G). Quantitation of HDAC5, AcK498-PARP1, and AcK521-PARP1 levels (H). Statistical analyses were done using one-way ANOVA (Dunn’s multiple comparison test). Error bars are means ± SD. ** P < 0.01; *** P < 0.001.
Article Snippet: HT29 cells were stably transfected (pooled neomycin-resistant population) with pGL4.51[ luc2 /CMV/Neo] plasmid (Promega) to generate HT29 Luc2 cells.
Techniques: Control, shRNA, Knockdown, Expressing, Injection, Imaging, Ex Vivo, Staining, Western Blot, Immunostaining, Quantitation Assay, Comparison
![( A ) HT29 <t>Luc2</t> control (Control; Luc shRNA); HDAC5 knockdown (HDAC5kd; HDAC5 shRNA); PARP1 knockdown (PARP1kd; PARP1 shRNA); PARP1 knockdown cells (PARP1kd; PARP1 shRNA) expressing PARP1 K498R (PARP1kd/PARP1 K498R ), PARP1 K521R (PARP1kd/PARP1 K521R ), or PARP1 K498R,521R (PARP1kd/PARP1 K498R,521R ); and HDAC5 knockdown cells expressing HDAC5 S259/498A (HDAC5kd/HDAC5 S259/498A ) were orthotopically injected into the cecum wall of nude mice. After 1 week, mice were administered 5FU (10 mg/kg) every alternate day. Olaparib was also coadministered at a dose of 10 mg/kg daily as indicated. Bioluminescence imaging was performed. ( B ) Bioluminescence quantification [(A) above]. The data are representative of three independent experiments using five individual mice per group. Error bars are means ± SD from five individual mice ( n = 5 mice per group). Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). *** P < 0.001. ( C and D ) At the end of 5 weeks, the liver and lung from indicated mice [(A) above] were analyzed by ex vivo imaging (C) and hematoxylin and eosin staining (D). ( E ) At the end of 5 weeks, the blood from mice in (A) above was used to isolate genomic DNA for examining circulating tumor cells. The data are representative of three independent experiments using five individual mice per group. Error bars are means ± SD from five individual mice ( n = 5 mice per group) * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) At the end of 5 weeks, lysates of primary orthotopic tumors from (A) above were analyzed by immunoblotting for the indicated proteins. ( G and H ) Representative image of immunostaining of the indicated proteins in different grades of colon adenocarcinoma and matched normal adjacent tissue (NAT) (G). Quantitation of HDAC5, AcK498-PARP1, and AcK521-PARP1 levels (H). Statistical analyses were done using one-way ANOVA (Dunn’s multiple comparison test). Error bars are means ± SD. ** P < 0.01; *** P < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8539/pmc11488539/pmc11488539__sciadv.ado7720-f7.jpg)