Journal: Arthritis Research & Therapy

Article Title: PARP-1 prevents osteoarthritis pathogenesis by inhibiting apoptosis in chondrocytes: an animal study

doi: 10.1186/s13075-026-03728-7

Figure Lengend Snippet: PARP-1 regulates cartilage degeneration in OA mice. a Genotypes, along with mRNA and protein expression levels of PARP-1, were evaluated in primary cultured chondrocytes isolated from WT and PARP-1 KO mice. b Immunostaining for PARP-1 was performed on the cartilage and synovium of WT and PARP-1 KO mice. Represented data of ( a )( b ) were collected from biological triplicate. c Cartilage degeneration, erosion, and subchondral bone sclerosis/osteophyte formation were evaluated by Safranin-O staining. Quantification of the OARSI grade (d) , osteophyte maturity ( e ), osteophyte size ( f ), and subchondral bone plate thickness ( g ) are presented. h Synovitis was analyzed using hematoxylin and eosin staining. i Quantification of synovitis is shown. WT ( n = 10) or PARP-1 KO ( n = 17) mice were used. All values, derived from the Mann–Whitney U test, are expressed as the mean ± standard error of the mean. Safranin O-stained images of the joint sections are presented, illustrating the whole joint (50X), subchondral bone sclerosis/osteophyte size (100X), and cartilage and synovium (200X). Scale bar: 50 μm. Abbreviations: WT, wild-type; KO, knockout; PARP-1, poly (ADP-ribose) polymerase-1; OARSI, Osteoarthritis Research Society International

Article Snippet: Eight-week-old to ten-week-old female 129 S PARP-1 knockout (KO) mice (strain number [no.] 002779; Jackson Laboratory, Bar Harbor, ME, USA) were used to investigate the mechanistic role of PARP-1 in OA progression.

Techniques: Expressing, Cell Culture, Isolation, Immunostaining, Staining, Derivative Assay, MANN-WHITNEY, Knock-Out

Journal: Arthritis Research & Therapy

Article Title: PARP-1 prevents osteoarthritis pathogenesis by inhibiting apoptosis in chondrocytes: an animal study

doi: 10.1186/s13075-026-03728-7

Figure Lengend Snippet: Expression of PARP-1 and its effects on cartilage degeneration in WT and PARP-1 KO. a Expression of PARP-1 was analyzed in cartilage and synovium tissue from WT and PARP-1 KO. Represented images were collected from biological triplicate. TUNEL assay was performed to assess apoptosis in the cartilage ( b ) and synovium ( d ) of WT and PARP-1 KO mice. Quantification of apoptotic cells in cartilage ( c , n = 7) and synovium ( e , n = 4) were presented. All values, derived from the two-tailed Student’s t-test, are expressed as the mean ± standard error of the mean. Scale bar: 50 μm. Abbreviations: WT, wild-type; KO, knockout; PARP-1, poly (ADP-ribose) polymerase-1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling

Article Snippet: Eight-week-old to ten-week-old female 129 S PARP-1 knockout (KO) mice (strain number [no.] 002779; Jackson Laboratory, Bar Harbor, ME, USA) were used to investigate the mechanistic role of PARP-1 in OA progression.

Techniques: Expressing, TUNEL Assay, Derivative Assay, Two Tailed Test, Knock-Out

Journal: Arthritis Research & Therapy

Article Title: PARP-1 prevents osteoarthritis pathogenesis by inhibiting apoptosis in chondrocytes: an animal study

doi: 10.1186/s13075-026-03728-7

Figure Lengend Snippet: Genetic or pharmaceutical inhibition of PARP-1 increases apoptosis in primary cultured chondrocytes. a MTT assay was performed to evaluate the effect of CHX and 3-AB on chondrocyte viability ( n = 8). Chondrocytes were treated with CHX (25–100 µM), and 3-AB (100 µM) for 24 h. TUNEL assay was performed in primary cultured chondrocytes treated with CHX (50 µM) and 3-AB (100 µM) ( b ), with quantification results presented ( c , n = 6). Western blot analysis was also performed ( d ). Flow cytometry assay with PI/Annexin V was performed in primary cultured chondrocytes treated with CHX (50 µM) and 3-AB (100 µM) ( e ), with quantification results presented ( f , n = 8). Also, PI/Annexin V assay was performed in primary cultured chondrocytes from WT and PARP-1 KO treated with CHX ( g ), and quantification results were shown ( h , n = 8). TUNEL assay was performed in primary cultured chondrocytes from WT and PARP-1 KO treated with CHX ( i ), and quantification results were shown ( j , n = 6). Protein levels of PAPR-1, PAR, and c-caspase-3 were analyzed by Western blotting ( k ). Inflammasome-related factors, including NLRP3, caspase-1, and IL-1β, were also assessed ( l ). Western blotting data were collected from biological triplicate. All values, derived from the two-tailed Student’s t-test, are expressed as the mean ± standard error of the mean. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CHX, cycloheximide; 3-AB, 3-aminobenzamide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type; KO, knockout; PARP-1, poly (ADP-ribose) polymerase-1; PAR, poly ADP-ribose; c-caspase-3, cleaved caspase-3; NLRP3, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3; IL-1β, interleukin-1 beta

Article Snippet: Eight-week-old to ten-week-old female 129 S PARP-1 knockout (KO) mice (strain number [no.] 002779; Jackson Laboratory, Bar Harbor, ME, USA) were used to investigate the mechanistic role of PARP-1 in OA progression.

Techniques: Inhibition, Cell Culture, MTT Assay, TUNEL Assay, Western Blot, Flow Cytometry, Annexin V Assay, Derivative Assay, Two Tailed Test, Knock-Out, Binding Assay

Journal: Arthritis Research & Therapy

Article Title: PARP-1 prevents osteoarthritis pathogenesis by inhibiting apoptosis in chondrocytes: an animal study

doi: 10.1186/s13075-026-03728-7

Figure Lengend Snippet: Effect of PARP-1 KO on inflammatory factor-induced expression of anabolic and catabolic factors in chondrocytes. WT and PARP-1 KO chondrocytes were treated with IL-1β ( a ), TNF-α ( b ), IL-6 ( c ), and LPS ( d ) for 24 h. The mRNA expression levels of Sox9, Col2a1, Aggrecan, Mmp3, Mmp13, and Adamts5 were analyzed using conventional RT-PCR, with GADPH serving as an internal control. Represented images were collected from biological triplicate. Abbreviations: WT, wild-type; KO, knockout; IL-1β, interleukin-1 beta; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; LPS, lipopolysaccharide; Sox9, SRY-box transcription factor 9; Col2a1, collagen type II alpha 1 chain; Aggrecan, aggrecan proteoglycan; Mmp3, matrix metalloproteinase 3; Mmp13, matrix metalloproteinase 13; Adamts5, a disintegrin and metalloproteinase with thrombospondin motifs 5; RT-PCR, reverse transcription polymerase chain reaction; and GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: Eight-week-old to ten-week-old female 129 S PARP-1 knockout (KO) mice (strain number [no.] 002779; Jackson Laboratory, Bar Harbor, ME, USA) were used to investigate the mechanistic role of PARP-1 in OA progression.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Knock-Out, Reverse Transcription, Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway

doi: 10.3389/fonc.2022.976292

Figure Lengend Snippet: KP372-1 enhances the lethality of PARP inhibitors in various cancer cells and depends on NQO1 activity. (A) PARP1 mRNA expression in matched pan-cancer tumor tissue. Data were from TCGA and analyzed with GEPIA web server. Red color indicates tumor sample; green indicates associated normal patient sample; blue color suggests up-regulation of PARP1 ; light blue indicates down-regulation of PARP1 ; black indicates normal expression of PARP1 . "*" (in red color) shows the representative cancer types we are focused of this article, which have normal PARP1 expression. (B, C) Representative IHC staining of PARP1 in breast (B) and lung (C) cancer patient samples or associated normal tissues. (D) Correlation between AKT1 and PARP1 in patient data obtained from TCGA. (E) Cell viability of combination treatment of KP372-1 with various PARP inhibitors in NSCLC A549 cells. (F, G) Cell viability of combination treatment of KP372-1 with rucaparib ± DIC in NSCLC A549 (F) and pancreatic cancer MiaPaCa-2 cells (G) . (H, I) Cell viability of combination treatment of KP372-1 ± rucaparib in NQO1 -knockout A549 (H) and MiaPaCa-2 (I) cell lines. (J, K) Cell viability of combination treatment of KP372-1 ± rucaparib in MDA-MB-231 (J) and stable NQO1 expressing MDA-MB-231 (K) cell lines. (E–K) Cells were pre-treated ± rucaparib or other PARP inhibitors for 2 h, then exposed to KP372-1 ± rucaparib or other PARP inhibitors for 2 h, followed by washing and replacing fresh media, Cell viability was determined by DNA assay 7 days later. Data are shown as mean ± SD, each experiment was done three independent times. Scale bar indicates 110 μm. (E–G) *** P < 0.001, comparing each data point with KP372-1 treatments ( t tests).

Article Snippet: Plasmid of PARP1 CRISPR/Cas9 knockout (sc-400046) was obtained from Santa Cruz.

Techniques: Activity Assay, Expressing, Immunohistochemistry, Knock-Out

Journal: Frontiers in Oncology

Article Title: KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway

doi: 10.3389/fonc.2022.976292

Figure Lengend Snippet: KP372-1 causes Ca 2+ releasing and AKT hyperactivation to enhance the lethality of PARP inhibitor. (A) Relative survival assay in A549 NSCLC cells treated with BAPTA (5 μM) under conditions of KP372-1 ± rucaparib. Cells were pre-treated ± rucaparib (15 µM) for 1 h, then added ± BAPTA (5 µM, 1 h), and then exposed to KP372-1 ± rucaparib ± BAPTA for 2 h, followed by washing and replacing media, cell viability was assessed 7 days later. (B, C) , PAR and γH2AX alterations were assessed and quantified in A549 NSCLC (B) and MCF-7 cells (C) . (D, E) A549 cells were pre-treated ± rucaparib (15 µM, 2 h), then exposed to KP372-1 ± rucaparib for 2 h, followed by washing and replacing media, cells were collected at indicated time points and assessed for: (D) Levels of pAKT s473 and total AKT (t-AKT), and bottom panel showed quantification of pAKT s473 ; (E) Fluorescence image of pAKT s473 . (F) Cells were treated as (B, C) then pAKT s473 levels alterations were assessed and quantified in A549 NSCLC and MCF-7 cells. (G) MCF-7 scramble and si AKT1/2 cells were treated as (D) then cells were collected at 2 h and relative H 2 O 2 in MCF-7 cells were determined. Results were separately repeated at least three times, AKT knockdown efficiency for (E) was confirmed by Western blot in (G) . All error bars are means ± SD. (A) *** P < 0.001, comparing each data point with KP372-1 alone treatments (grey color) ( t tests). (F) , *** P < 0.001 and ns: no significant, comparing each group with control (DMSO) treatments ( t tests).

Article Snippet: Plasmid of PARP1 CRISPR/Cas9 knockout (sc-400046) was obtained from Santa Cruz.

Techniques: Clonogenic Cell Survival Assay, Fluorescence, Knockdown, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway

doi: 10.3389/fonc.2022.976292

Figure Lengend Snippet: PARP Inhibition blocks KP372-1-induced PARP1 hyperactivation and amplifies DNA damage. (A) A549 cells were pre-treated ± rucaparib (15 µM, 2 h), then exposed to supra-lethal dose of KP372-1 (0.4 µM) ± rucaparib for indicated times, then PAR, γH2AX and NQO1 expression alterations were assessed and quantified. (B) A549 PARP1 knockout cells were exposed to ± KP372-1 (0.4 µM) for 5 -120 min, western blot analysis of PAR and γH2AX formation at indicated time points. (C) A549 cells were pre-treated ± rucaparib (15 µM, 2 h), then exposed to KP372-1 (0.2 or 0.4 µM) ± rucaparib (added at t = 20 min, arrow), and real-time oxygen consumption rates (OCRs) were assessed by Seahorse XF analyses. Oligo, oligomycin. (D) MCF-7 cells were pre-treated ± rucaparib (0.4 μM, 2 h), then exposed to KP372-1 (0.08 or 0.4 µM) ± rucaparib for 2 h, relative H 2 O 2 levels were assessed. (E, F) cells were pre-treated ± rucaparib (2 h), then exposed to rucaparib ± KP372-1 for 2 h, relative H 2 O 2 levels were assessed in A549 PARP1 -KO (E) and NQO1 -KO (F) cells, (G, H) A549 cells were pre-treated ± rucaparib (2 h), then exposed to rucaparib ± KP372-1, cells were collected at indicated time points and assessed for: (G) Comet tail-lengths determined by alkaline comet assays; (H) DNA double strand breaks (DSBs) indicated by γH2AX with immunofluorescence staining. All error bars are means ± SD from three independent experiments. Scale bar indicates 10 µm. (D–F) *** P < 0.001 and ns: no significant, comparing each group or each data point with control (DMSO) treatments ( t tests).

Article Snippet: Plasmid of PARP1 CRISPR/Cas9 knockout (sc-400046) was obtained from Santa Cruz.

Techniques: Inhibition, Expressing, Knock-Out, Western Blot, Immunofluorescence, Staining, Control

Journal: Frontiers in Oncology

Article Title: KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway

doi: 10.3389/fonc.2022.976292

Figure Lengend Snippet: KP372-1 inhibits DNA repair to overcome resistance of PARP inhibitor. (A) A549 NSCLC cells were treated with rucaparib (15 µM or 50 µM) for 4 h, followed by washing and replacing fresh media, then cells were collected at indicated times and assessed for: pAKT s473 , FOXO3a, and GADD45α alterations. (B–G) Cells were pre-treated ± rucaparib (15 μM, 2h), then exposed to KP372-1 ± rucaparib (15 μM) for 2 h followed by washing and replacing media, cells were collected at indicated timepoints and assessed for: (B) FOXO3a and GADD45α levels in A549 cells; (C) pAKT s473 , t-AKT, FOXO3a, GADD45α, and γH2AX alterations in A549 scramble and si AKT1/2 cells; (D) Fluorescence images of FOXO3a alterations in A549 scramble and si AKT1/2 cells; (E) Quantification of RAD51 foci per nuclei in A549 scramble and si AKT1/2 cells; (F) , Immunofluorescence staining of RAD51 expression in A549 scramble and si AKT1/2 cells; (G, H) The expression and quantification of pAKT s473 in A549 scramble and si FOXO3a cells. (I) A549 scramble and si FOXO3a cells were pre-treated ± rucaparib (15 μM, 2h), then exposed to KP372-1 ± rucaparib (15 μM) for 2 h followed by washing and replacing media, cell viability was assessed after 7 days. The efficiency of FOXO3a knockdown was confirmed by Western blot analysis. All results were separately repeated at least three times. AKT knockdown efficiency for (D–F) was confirmed by Western blot shown in (C) . Scale bar indicates 25 µm (D) and 15 µm (E) respectively. Error bars are means ± SD. (F) * P < 0.05, ** P < 0.01 and *** P < 0.001, comparing each group with control (DMSO) treatments ( t tests). (H) ** P < 0.01, comparing each data point with KP372-1+ rucaparib treatments in A549 scramble cells (red color) ( t tests).

Article Snippet: Plasmid of PARP1 CRISPR/Cas9 knockout (sc-400046) was obtained from Santa Cruz.

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

Journal: Frontiers in Oncology

Article Title: KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway

doi: 10.3389/fonc.2022.976292

Figure Lengend Snippet: Combination of KP372-1 with PARP inhibitor induces cell autophagy and cell apoptosis. Cells were pre-treated ± rucaparib (0.4 µM or 15 µM, 2 h), then exposed to KP372-1 ± rucaparib or KP372-1 ± rucaparib ± Bafilomycin A1 (Baf A1) for 2 h followed by washing and replacing media; cells treated with Baf A1 (0.05 µM) were kept with Baf A1 for 24 h; positive control cells were exposed to Staurosporine (STS, 1 μM) for 18 h; then cells including debris in media were finally collected at indicated timepoints and examined for: (A) Annexin-V/7-AAD staining to determine cell death way via flow cytometry, early apoptosis part indicated by Annexin-V + /7-AAD - was quantified on the left pannel; (B) PARP1 and cleaved caspase 7 alterations in A549 cells; (C) Levels of cleaved caspase 7 in MCF-7 cells; (D, E) LC3 I/II and p62 levels in A549 cells. Results were separately repeated at least three times and protein levels were quantified. (A) Error bars are means ± SD. ** P < 0.01 *** P < 0.001, comparing each group with control (DMSO) treatment ( t tests).

Article Snippet: Plasmid of PARP1 CRISPR/Cas9 knockout (sc-400046) was obtained from Santa Cruz.

Techniques: Positive Control, Staining, Flow Cytometry, Control

Journal: Frontiers in Oncology

Article Title: KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway

doi: 10.3389/fonc.2022.976292

Figure Lengend Snippet: KP372-1 synergizes with PARP inhibitor against orthotopic A549 and NQO1 -KO NSCLC xenografts. (A–C) Orthotopic A549 and NQO1 knockout (A549 NQO1 -KO) tumors were established in 20-22 g female NSG mice by injecting 1 x 10 6 (A549) or 1.1 x 10 6 (A549 NQO1 -KO) cells/mouse into lung via intravenous tail vein. After two weeks, mice were treated with/without rucaparib (10 mg/kg, i.p.) for 2 h followed by HPβCD (Vehicle) or HPβCD-KP372-1 (KP372-1) (16 mg/kg, i.v.) every other day for 5 injections. Experiments were repeated at least two times, n = 5/group. Images of representative mouse tumors at indicated times and quantified tumor volumes (right panel). (B) Kaplan-Meier survival curves of A549 orthotopic mice. (C) Kaplan-Meier survival curves of A549 NQO1 -KO orthotopic mice. (D) Orthotopic A549 tumor-bearing female NSG mice ( n = 3/group) were treated as in (A) and sacrificed at 2 h, relative NAD + levels of tumor tissues were determined. (E) Pharmacokinetics (PK) of KP372-1 in orthotopic A549 tumor-bearing female NSG mice ( n = 3/group) treated as in (A) and sacrificed at indicated times. (F) summary description of this study. Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, ns: no significant, comparing each data point with those of vehicle treatments determined by unpaired Student’s t -test (A, D, E) or log-rank test in (B, C) . (B) Synergy values (η = 0.82) were reported based on multiple dose responses, or on comparative p values indicated.

Article Snippet: Plasmid of PARP1 CRISPR/Cas9 knockout (sc-400046) was obtained from Santa Cruz.

Techniques: Knock-Out, Drug discovery