dna stain dapi  (Thermo Fisher)

Journal: bioRxiv

Article Title: PARP16 is a Druggable Regulator of Ribosome MARylation and Protein Homeostasis in Ovarian Cancer Cells

doi: 10.64898/2026.04.01.715986

Figure Lengend Snippet: (A and B) DB008 engages PARP16 and reduces its MARylation in ovarian cancer cells. OVCAR3 cells ectopically expressing FLAG-PARP16 were treated with 0 – 10 µM DB008 for 4 hours. (A) 20 µg of lysate were subjected to click chemistry to visualize DB008 target engagement with in-gel fluorescence (arrow indicates expected band size for FLAG-PARP16). (B) FLAG-PARP16 was immunoprecipitated from the remaining lysate and its MARylation was assessed with Western blotting. (C and D) In situ detection of (C) PARP16 autoMARylation and (D) RPS6 transMARylation. PLA of FLAG and MAR in OVCAR3 cells subjected to Dox-induced expression of FLAG-PARP16 and treated with 0 or 1 µM DB008 for 16 hours (C), or RPS6 and MAR in OVCAR3 cells treated with 0 or 1 µM DB008 for 16 hours (D). DNA was stained with DAPI. Scale bar is 25 µm. (E and F) Quantification of PLA foci in panel C (E) and panel D (F). Each bar represents the mean + SEM; n = 3. Welch’s t-test; ** = p < 0.01. (G) DB008 reduces ribosome MARylation. OVCAR3 cells were treated with 0 or 1 µM DB008 for 16 hours. Ribosomes were fractionated by ultracentrifugation and ribosome MARylation was assessed by Western blotting. RPS6 was used as a loading control. (H and I) DB008 enhances protein synthesis. OVCAR3 cells were treated with 0 – 2 µM DB008 for 16 hours. The rate of protein synthesis was then assessed with a puromycin incorporation assay. (D) Western blot showing puromycin incorporation, COX20, and cleaved caspase-3. β-tubulin was used as a loading control. (E) Quantification of puromycin signal in D. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different from the control; ANOVA; * = p < 0.05, **** = p < 0.0001.

Article Snippet: The stained cells were mounted on microslides using VectaShield Antifade Mounting Medium (Vector laboratories, H-1200-10) with DAPI DNA stain.

Techniques: Expressing, Drug discovery, Fluorescence, Immunoprecipitation, Western Blot, In Situ, Staining, Control

Journal: bioRxiv

Article Title: PARP16 is a Druggable Regulator of Ribosome MARylation and Protein Homeostasis in Ovarian Cancer Cells

doi: 10.64898/2026.04.01.715986

Figure Lengend Snippet: (A) Western blot showing PARP16 protein expression is abolished in OVCAR3 cells with PARP16 KO. β-actin was used as a loading control. (B) Immunofluorescence with the MAR detection reagent in OVCAR3 Control or PARP16 KO cells treated with 0 or 1 µM DB008 for 16 hours. The DNA was stained with DAPI. Scale bar = 25 µm. (C) The DB008-dependent increase in protein synthesis requires PARP16 expression. OVCAR3 Control or PARP16 KO cells were treated with 0 or 1 µM DB008 for 16 hours. Protein synthesis was then assessed with a puromycin incorporation assay. β-tubulin was used as a loading control. (D) The DB008-dependent increase in protein aggregation requires PARP16 expression. OVCAR3 Control or PARP16 KO cells were treated with 0 or 1 µM DB008 for 16 hours. Protein aggregation was then assessed with the proteostat aggresome detection reagent. The DNA was stained with DAPI. Scale bar = 25 µm. (E) DB008 reduces growth of control but not OVCAR3 PARP16 KO cells. Cells were treated with 0 or 1 µM DB008 for 1 week. Cell growth was then determined with crystal violet staining. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different from the control; ANOVA; ** = p < 0.005; ns indicates no significance. (F) DB008 inhibits the anchorage-independent growth of ovarian cancer cells. OVCAR3 cells were grown in soft agar with media containing 0 – 1 µM DB008 for 3 weeks, replenishing media once per week. Anchorage-independent growth was then measured with crystal violet staining. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different from the control; ANOVA; * = p < 0.05, **** = p < 0.0001.

Article Snippet: The stained cells were mounted on microslides using VectaShield Antifade Mounting Medium (Vector laboratories, H-1200-10) with DAPI DNA stain.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: PARP16 is a Druggable Regulator of Ribosome MARylation and Protein Homeostasis in Ovarian Cancer Cells

doi: 10.64898/2026.04.01.715986

Figure Lengend Snippet: (A and B) Inhibition by DB008 requires PARP16 C169. (A) IP-Western showing PARP16 MARylation ( top panel ) or PARylation levels in whole cell extracts (WCE; bottom panel ) of OVCAR3 PARP16 KO cells stably expressing FLAG-PARP16 or FLAG-PARP16 C169S after treatment with 1 µM DB008 for 4 hours. (B) Quantification of MAR signal from A. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different from the control; ANOVA; * = p < 0.05; ns indicates no significance. (C) Anchorage-independent growth is unaffected in cells expressing PARP16 C169S. OVCAR3 PARP16 KO cells stably expressing PARP16 WT or PARP16 C169S were grown in soft agar with media containing 0 or 1 µM DB008 for 3 weeks, replenishing media once per week. Anchorage-independent growth was then measured with crystal violet staining. Quantification of crystal violet absorbance in B. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different from the control; ANOVA; * = p < 0.05; ns indicates no significance. (D and E) The DB008-dependent increase in protein synthesis requires PARP16 C169. (D) OVCAR3 PARP16 KO cells stably expressing PARP16 WT or PARP16 C169S were treated with 0 or 1 µM DB008 for 16 hours. Protein synthesis was then assessed with a puromycin incorporation assay. β-tubulin was used as a loading control. (E) Quantification of puromycin signal in D. Each bar represents the mean + SEM; n = 3. Bars marked with asterisks are significantly different from the control; ANOVA; ** = p < 0.01; ns indicates no significance. (F) The DB008-dependent increase in protein aggregation requires PARP16 C169. OVCAR3 PARP16 KO cells stably expressing PARP16 WT or PARP16 C169S were treated with 0 or 1 µM DB008 for 16 hours. Protein aggregation was then assessed with the proteostat aggresome detection reagent. The DNA was stained with DAPI. Scale bar = 25 µm.

Article Snippet: The stained cells were mounted on microslides using VectaShield Antifade Mounting Medium (Vector laboratories, H-1200-10) with DAPI DNA stain.

Techniques: Inhibition, Western Blot, Stable Transfection, Expressing, Control, Staining