Journal: Biochimica et biophysica acta

Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

doi: 10.1016/j.bbadis.2017.04.024

Figure Lengend Snippet: Effect of PARP-1 on the glucose- induced MMP-9 expression: Gene transcripts of (a) MMP-9 and (b) PARP-1 were quantified in the cells incubated in normal or high glucose in the presence of PJ34 or PARP-1 siRNA or scrambled siRNA by qPCR. β-actin was used as the housekeeping gene. (c) Nuclear localization of PARP-1 was determined by immunofluorescence using Alexa-Flour 488 (green) conjugated secondary antibody. The cover slips were mounted using DAPI-containing mounting medium (blue). Each measurement was made in duplicate in 4–5 samples in each group, and the values are represented as mean ± SD. 5mM and 20mM = cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si-P and C = cells incubated with PJ34, PARP-1 siRNA, or scrambled siRNA respectively, and incubated in 20mM glucose. *P<0.05 versus 5mM glucose and #P<0.05 versus 20mM glucose.

Article Snippet: The results were normalized to the expression of β-actin or 18S rRNA, and the relative fold change in the expression was calculated using the delta delta Ct method [ 10 , 27 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Sequence Human MMP- 9 promoter, NF- k B binding site 5′-GATTCAGCCTGCGGAAGACAGGG-3′ 5′-CCAAACCCCTCCCCACACTCCA-3′ MMP-9 promoter, AP-1 proximal site 5′-GAGTCAGCACTTGCCTGTCA-3′ 5′-CTGCTGTTGTGGGGGCTTTA-3′ MMP-9 promoter, AP-1 distal site 5′-CTTGCCTAGCAGAGCCCATT-3′ 5′-TTTTTCCCTCCCTGACAGCC-3′ PARP-1 5′-GCTTCAGCCTCCTTGCTACA-3′ 5′-TTCGCCACTTCATCCACTCC-3′ MMP-9 5′-CACTGTCCACCCCTCAGAGC-3′ 5′-GCCACTTGTCGGCGATAAGG-3′ Cytb 5′-TCACCAGACGCCTCAACCGC-3′ 5′-GCCTCGCCCGATGTGTAGGA-3′ β-Actin 5′-AGCCTCGCCTTTGCCGATCCG-3′ 5′-TCTCTTGCTCTGGGCCTCGTCG-3′ Mouse MMP- 9 promoter, NF- k B binding site 5′-GCCCCATGGAATTCCCCAAA-3′ 5′-CCGCCCCCTGATAGAGTCTT-3′ MMP-9 promoter, AP-1 proximal site 5′-CAGGGCCTCGTCTTTCTTTC-3′ 5′-CCATGGTTTGGTGTTGCTGTT-3′ MMP-9 promoter, AP-1 distal site 5′-AGCGCCAGTTCTGTTAGCAT-3′ 5′-TAGACGTCCACGAGTCTGGG-3′ MMP-9 5′-GGGGTTTGCCCCATGGAAT-3′ 5′-GAGCCCATCCCCACACTGTA-3′ 18S 5′-GCCCTGTAATTGGAATGAGTCCACTT-3′ 5′-CTCCCCAAGATCCAACTACGAGCTTT-3′ Open in a separate window Primer sequence Nuclear localization of PARP-1 was investigated in retinal endothelial cells by immunofluorescence technique using PARP-1 primary antibody (Catalog No: sc-7150, Santa Cruz Biotechnology) and Alexa Fluor-488 conjugated anti-rabbit secondary antibody (green; Molecular Probes-Life Technologies, Grand Island, NE).

Techniques: Expressing, Incubation, Immunofluorescence

Journal: Biochimica et biophysica acta

Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

doi: 10.1016/j.bbadis.2017.04.024

Figure Lengend Snippet: Regulation of PARP-1 and binding of NF-kB and AP-1 at the MMP-9 promoter: Endothelial cells incubated with PJ34 or PARP-1 siRNA, were analyzed for binding of (a) NF-kB, and (b) AP-1 at the proximal and the distal regions of MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). Each measurement was made in duplicate in 3–4 samples/group. 5mM and 20mM=cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si-P and C = cells incubated in 20mM glucose in the presence of PJ34, PARP-1 siRNA, and scrambled siRNA control respectively. *P<0.05 versus 5mM glucose and #P<0.05 versus 20mM glucose.

Article Snippet: The results were normalized to the expression of β-actin or 18S rRNA, and the relative fold change in the expression was calculated using the delta delta Ct method [ 10 , 27 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Sequence Human MMP- 9 promoter, NF- k B binding site 5′-GATTCAGCCTGCGGAAGACAGGG-3′ 5′-CCAAACCCCTCCCCACACTCCA-3′ MMP-9 promoter, AP-1 proximal site 5′-GAGTCAGCACTTGCCTGTCA-3′ 5′-CTGCTGTTGTGGGGGCTTTA-3′ MMP-9 promoter, AP-1 distal site 5′-CTTGCCTAGCAGAGCCCATT-3′ 5′-TTTTTCCCTCCCTGACAGCC-3′ PARP-1 5′-GCTTCAGCCTCCTTGCTACA-3′ 5′-TTCGCCACTTCATCCACTCC-3′ MMP-9 5′-CACTGTCCACCCCTCAGAGC-3′ 5′-GCCACTTGTCGGCGATAAGG-3′ Cytb 5′-TCACCAGACGCCTCAACCGC-3′ 5′-GCCTCGCCCGATGTGTAGGA-3′ β-Actin 5′-AGCCTCGCCTTTGCCGATCCG-3′ 5′-TCTCTTGCTCTGGGCCTCGTCG-3′ Mouse MMP- 9 promoter, NF- k B binding site 5′-GCCCCATGGAATTCCCCAAA-3′ 5′-CCGCCCCCTGATAGAGTCTT-3′ MMP-9 promoter, AP-1 proximal site 5′-CAGGGCCTCGTCTTTCTTTC-3′ 5′-CCATGGTTTGGTGTTGCTGTT-3′ MMP-9 promoter, AP-1 distal site 5′-AGCGCCAGTTCTGTTAGCAT-3′ 5′-TAGACGTCCACGAGTCTGGG-3′ MMP-9 5′-GGGGTTTGCCCCATGGAAT-3′ 5′-GAGCCCATCCCCACACTGTA-3′ 18S 5′-GCCCTGTAATTGGAATGAGTCCACTT-3′ 5′-CTCCCCAAGATCCAACTACGAGCTTT-3′ Open in a separate window Primer sequence Nuclear localization of PARP-1 was investigated in retinal endothelial cells by immunofluorescence technique using PARP-1 primary antibody (Catalog No: sc-7150, Santa Cruz Biotechnology) and Alexa Fluor-488 conjugated anti-rabbit secondary antibody (green; Molecular Probes-Life Technologies, Grand Island, NE).

Techniques: Binding Assay, Incubation

Journal: Biochimica et biophysica acta

Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

doi: 10.1016/j.bbadis.2017.04.024

Figure Lengend Snippet: Regulation of PARP-1 and its effect on mtDNA damage and cell apoptosis: Effect of PARP-1 regulation on (a) mtDNA-encoded Cytb expression was measured by qPCR using β-actin as the housekeeping gene, and (b) apoptosis by an ELISA kit for histone-associated-DNA-fragments in the cells incubated with PJ34 or PARP-1 siRNA, or scrambled siRNA control. The values are represented as mean ± SD from 3–4 samples/group. 5mM and 20mM=cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si-P and C = cells incubated in 20mM glucose in the presence of PJ34, PARP-1 siRNA and scrambled siRNA control, respectively. *P<0.05 versus 5mM glucose and #P<0.05 versus 20mM glucose.

Article Snippet: The results were normalized to the expression of β-actin or 18S rRNA, and the relative fold change in the expression was calculated using the delta delta Ct method [ 10 , 27 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Sequence Human MMP- 9 promoter, NF- k B binding site 5′-GATTCAGCCTGCGGAAGACAGGG-3′ 5′-CCAAACCCCTCCCCACACTCCA-3′ MMP-9 promoter, AP-1 proximal site 5′-GAGTCAGCACTTGCCTGTCA-3′ 5′-CTGCTGTTGTGGGGGCTTTA-3′ MMP-9 promoter, AP-1 distal site 5′-CTTGCCTAGCAGAGCCCATT-3′ 5′-TTTTTCCCTCCCTGACAGCC-3′ PARP-1 5′-GCTTCAGCCTCCTTGCTACA-3′ 5′-TTCGCCACTTCATCCACTCC-3′ MMP-9 5′-CACTGTCCACCCCTCAGAGC-3′ 5′-GCCACTTGTCGGCGATAAGG-3′ Cytb 5′-TCACCAGACGCCTCAACCGC-3′ 5′-GCCTCGCCCGATGTGTAGGA-3′ β-Actin 5′-AGCCTCGCCTTTGCCGATCCG-3′ 5′-TCTCTTGCTCTGGGCCTCGTCG-3′ Mouse MMP- 9 promoter, NF- k B binding site 5′-GCCCCATGGAATTCCCCAAA-3′ 5′-CCGCCCCCTGATAGAGTCTT-3′ MMP-9 promoter, AP-1 proximal site 5′-CAGGGCCTCGTCTTTCTTTC-3′ 5′-CCATGGTTTGGTGTTGCTGTT-3′ MMP-9 promoter, AP-1 distal site 5′-AGCGCCAGTTCTGTTAGCAT-3′ 5′-TAGACGTCCACGAGTCTGGG-3′ MMP-9 5′-GGGGTTTGCCCCATGGAAT-3′ 5′-GAGCCCATCCCCACACTGTA-3′ 18S 5′-GCCCTGTAATTGGAATGAGTCCACTT-3′ 5′-CTCCCCAAGATCCAACTACGAGCTTT-3′ Open in a separate window Primer sequence Nuclear localization of PARP-1 was investigated in retinal endothelial cells by immunofluorescence technique using PARP-1 primary antibody (Catalog No: sc-7150, Santa Cruz Biotechnology) and Alexa Fluor-488 conjugated anti-rabbit secondary antibody (green; Molecular Probes-Life Technologies, Grand Island, NE).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Biochimica et biophysica acta

Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

doi: 10.1016/j.bbadis.2017.04.024

Figure Lengend Snippet: Effect of Sirt1 regulation on PARP-1 binding: Endothelial cells transfected with Sirt1 cDNA and incubated in 20mM glucose, were analyzed for the binding of PARP-1 at (a) NF-kB and (b) AP-1 binding regions of the MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). (c) PARP-1 acetylation and its effect on the binding of NF-kB/AP-1 with PARP-1 were determined by immunoprecipitating total proteins using PARP-1 antibody, followed by western blotting for NF-kB/AP-1. The values are represented as mean ± SD from 3–4 samples/group. 5mM and 20mM = cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+St and 20+R = cells transfected with Sirt1 plasmids or transfection reagent alone respectively, and incubated in 20mM glucose. *P<0.05 versus 5mM glucose and #P<0.05 versus 20mM glucose.

Article Snippet: The results were normalized to the expression of β-actin or 18S rRNA, and the relative fold change in the expression was calculated using the delta delta Ct method [ 10 , 27 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Sequence Human MMP- 9 promoter, NF- k B binding site 5′-GATTCAGCCTGCGGAAGACAGGG-3′ 5′-CCAAACCCCTCCCCACACTCCA-3′ MMP-9 promoter, AP-1 proximal site 5′-GAGTCAGCACTTGCCTGTCA-3′ 5′-CTGCTGTTGTGGGGGCTTTA-3′ MMP-9 promoter, AP-1 distal site 5′-CTTGCCTAGCAGAGCCCATT-3′ 5′-TTTTTCCCTCCCTGACAGCC-3′ PARP-1 5′-GCTTCAGCCTCCTTGCTACA-3′ 5′-TTCGCCACTTCATCCACTCC-3′ MMP-9 5′-CACTGTCCACCCCTCAGAGC-3′ 5′-GCCACTTGTCGGCGATAAGG-3′ Cytb 5′-TCACCAGACGCCTCAACCGC-3′ 5′-GCCTCGCCCGATGTGTAGGA-3′ β-Actin 5′-AGCCTCGCCTTTGCCGATCCG-3′ 5′-TCTCTTGCTCTGGGCCTCGTCG-3′ Mouse MMP- 9 promoter, NF- k B binding site 5′-GCCCCATGGAATTCCCCAAA-3′ 5′-CCGCCCCCTGATAGAGTCTT-3′ MMP-9 promoter, AP-1 proximal site 5′-CAGGGCCTCGTCTTTCTTTC-3′ 5′-CCATGGTTTGGTGTTGCTGTT-3′ MMP-9 promoter, AP-1 distal site 5′-AGCGCCAGTTCTGTTAGCAT-3′ 5′-TAGACGTCCACGAGTCTGGG-3′ MMP-9 5′-GGGGTTTGCCCCATGGAAT-3′ 5′-GAGCCCATCCCCACACTGTA-3′ 18S 5′-GCCCTGTAATTGGAATGAGTCCACTT-3′ 5′-CTCCCCAAGATCCAACTACGAGCTTT-3′ Open in a separate window Primer sequence Nuclear localization of PARP-1 was investigated in retinal endothelial cells by immunofluorescence technique using PARP-1 primary antibody (Catalog No: sc-7150, Santa Cruz Biotechnology) and Alexa Fluor-488 conjugated anti-rabbit secondary antibody (green; Molecular Probes-Life Technologies, Grand Island, NE).

Techniques: Binding Assay, Transfection, Incubation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

doi: 10.1016/j.bbadis.2017.04.024

Figure Lengend Snippet: Effect of PARP-1 on diabetes-induced NF-kB/AP-1 binding at MMP-9 promoter in retinal microvessels: Binding of (a) NF-kB, and (b) AP-1 at MMP-9 promoter was measured in retinal microvessels using NF-kB and AP-1 antibodies. IgG was used as an antibody control (indicated as ^). (c) Retinal microvasculature from diabetic mice receiving PJ34 was quantified for MMP-9 expression by qPCR using 18S as the housekeeping gene. The values are represented as mean ± SD from 4–6 mice/group. Nor and Dia = C57BL/6J mice normal and diabetic respectively, and Dia+PJ34 = C57BL/6J diabetic mice administered with PJ34. *P<0.05 compared to Nor and #P<0.05 compared to diabetes.

Article Snippet: The results were normalized to the expression of β-actin or 18S rRNA, and the relative fold change in the expression was calculated using the delta delta Ct method [ 10 , 27 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Sequence Human MMP- 9 promoter, NF- k B binding site 5′-GATTCAGCCTGCGGAAGACAGGG-3′ 5′-CCAAACCCCTCCCCACACTCCA-3′ MMP-9 promoter, AP-1 proximal site 5′-GAGTCAGCACTTGCCTGTCA-3′ 5′-CTGCTGTTGTGGGGGCTTTA-3′ MMP-9 promoter, AP-1 distal site 5′-CTTGCCTAGCAGAGCCCATT-3′ 5′-TTTTTCCCTCCCTGACAGCC-3′ PARP-1 5′-GCTTCAGCCTCCTTGCTACA-3′ 5′-TTCGCCACTTCATCCACTCC-3′ MMP-9 5′-CACTGTCCACCCCTCAGAGC-3′ 5′-GCCACTTGTCGGCGATAAGG-3′ Cytb 5′-TCACCAGACGCCTCAACCGC-3′ 5′-GCCTCGCCCGATGTGTAGGA-3′ β-Actin 5′-AGCCTCGCCTTTGCCGATCCG-3′ 5′-TCTCTTGCTCTGGGCCTCGTCG-3′ Mouse MMP- 9 promoter, NF- k B binding site 5′-GCCCCATGGAATTCCCCAAA-3′ 5′-CCGCCCCCTGATAGAGTCTT-3′ MMP-9 promoter, AP-1 proximal site 5′-CAGGGCCTCGTCTTTCTTTC-3′ 5′-CCATGGTTTGGTGTTGCTGTT-3′ MMP-9 promoter, AP-1 distal site 5′-AGCGCCAGTTCTGTTAGCAT-3′ 5′-TAGACGTCCACGAGTCTGGG-3′ MMP-9 5′-GGGGTTTGCCCCATGGAAT-3′ 5′-GAGCCCATCCCCACACTGTA-3′ 18S 5′-GCCCTGTAATTGGAATGAGTCCACTT-3′ 5′-CTCCCCAAGATCCAACTACGAGCTTT-3′ Open in a separate window Primer sequence Nuclear localization of PARP-1 was investigated in retinal endothelial cells by immunofluorescence technique using PARP-1 primary antibody (Catalog No: sc-7150, Santa Cruz Biotechnology) and Alexa Fluor-488 conjugated anti-rabbit secondary antibody (green; Molecular Probes-Life Technologies, Grand Island, NE).

Techniques: Binding Assay, Expressing

Journal: Biochimica et biophysica acta

Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

doi: 10.1016/j.bbadis.2017.04.024

Figure Lengend Snippet: Diabetes-induced PARP-1 binding at MMP-9 promoter in retinal microvessels, and its regulation by Sirt1: DNA isolated from crosslinked retinal microvessels from Sirt1 overexpressing mice was analyzed for the binding of PARP-1 at (a) NF-kB and (b) AP-1 binding regions of the MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). (c) Effect of acetylation on the binding of NF-kB and of AP-1 with PARP-1 was determined by immunoprecipitating total proteins using PARP-1 antibody, followed by western blotting for NF-kB (p65) and of AP-1 (c-Jun). The values are represented as mean ± SD from 4–5 samples/group. Nor and Dia = C57BL/6J mice normal and diabetic respectively, and St-Nor and St-Dia = Sirt1 overexpressing mice normal and diabetic respectively. *P<0.05 compared to Nor and #P<0.05 compared to diabetes.

Article Snippet: The results were normalized to the expression of β-actin or 18S rRNA, and the relative fold change in the expression was calculated using the delta delta Ct method [ 10 , 27 ]. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Sequence Human MMP- 9 promoter, NF- k B binding site 5′-GATTCAGCCTGCGGAAGACAGGG-3′ 5′-CCAAACCCCTCCCCACACTCCA-3′ MMP-9 promoter, AP-1 proximal site 5′-GAGTCAGCACTTGCCTGTCA-3′ 5′-CTGCTGTTGTGGGGGCTTTA-3′ MMP-9 promoter, AP-1 distal site 5′-CTTGCCTAGCAGAGCCCATT-3′ 5′-TTTTTCCCTCCCTGACAGCC-3′ PARP-1 5′-GCTTCAGCCTCCTTGCTACA-3′ 5′-TTCGCCACTTCATCCACTCC-3′ MMP-9 5′-CACTGTCCACCCCTCAGAGC-3′ 5′-GCCACTTGTCGGCGATAAGG-3′ Cytb 5′-TCACCAGACGCCTCAACCGC-3′ 5′-GCCTCGCCCGATGTGTAGGA-3′ β-Actin 5′-AGCCTCGCCTTTGCCGATCCG-3′ 5′-TCTCTTGCTCTGGGCCTCGTCG-3′ Mouse MMP- 9 promoter, NF- k B binding site 5′-GCCCCATGGAATTCCCCAAA-3′ 5′-CCGCCCCCTGATAGAGTCTT-3′ MMP-9 promoter, AP-1 proximal site 5′-CAGGGCCTCGTCTTTCTTTC-3′ 5′-CCATGGTTTGGTGTTGCTGTT-3′ MMP-9 promoter, AP-1 distal site 5′-AGCGCCAGTTCTGTTAGCAT-3′ 5′-TAGACGTCCACGAGTCTGGG-3′ MMP-9 5′-GGGGTTTGCCCCATGGAAT-3′ 5′-GAGCCCATCCCCACACTGTA-3′ 18S 5′-GCCCTGTAATTGGAATGAGTCCACTT-3′ 5′-CTCCCCAAGATCCAACTACGAGCTTT-3′ Open in a separate window Primer sequence Nuclear localization of PARP-1 was investigated in retinal endothelial cells by immunofluorescence technique using PARP-1 primary antibody (Catalog No: sc-7150, Santa Cruz Biotechnology) and Alexa Fluor-488 conjugated anti-rabbit secondary antibody (green; Molecular Probes-Life Technologies, Grand Island, NE).

Techniques: Binding Assay, Isolation, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin induced apoptosis in both TNBC and NSCLC cells (A) TNBC cells (HCC70, MDA-MB-468, and BT549) and NSCLC cells (H460, H1650, and A549) were plated onto 6 cm dishes with culture medium containing 10% FBS. After 24 h, cells were treated with indicated concentrations of metformin in fresh medium with 5% FBS for 48 h. Both adherent and non-adherent cells were collected and subjected to apoptosis analysis using a cell death detection ELISA. Data show a representative of three independent experiments. Bars, SD. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) The same batch of cell lysates was used for western blot analyses with specific antibodies directed against PARP, caspase-8, caspase-3, or β-actin.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Inhibition of TRAIL function with a recombinant TRAIL-R2 Fc chimera significantly attenuated metformin-induced apoptosis in TNBC and NSCLC cells (A) TNBC and NSCLC cells were plated onto 6 cm dishes with medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium with 5% FBS containing either metformin or TRAIL-R2 Fc chimera protein alone or combinations of metformin and TRAIL-R2 Fc chimera for 48 h. Both adherent and non-adherent cells were collected and subjected to apoptosis ELISA. Data show a representative of three independent experiments. Bars, SD. ns, not significant, ∗p < 0.05, ∗∗p < 0.01. (B) The same batch of cell lysates was used for western blot analyses with specific antibodies directed against PARP, caspase-8, caspase-3, or β-actin.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Inhibition, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Specific knockdown of TRAIL expression inhibited metformin-induced apoptosis in TNBC and NSCLC cells TNBC and NSCLC cells were infected with lentivirus containing either control shRNA (sh-scr) or specific TRAIL -targeting shRNA (sh-1 or sh-2). The infected cells were selected by puromycin for 24 h and then treated with or without metformin for additional 48 h. (A) Both adherent and non-adherent cells were collected and subjected to western blot analyses of TRAIL, PARP, caspase-8, caspase-3, or β-actin. The densitometry analyses of TRAIL signals were shown underneath, and the arbitrary numbers indicated the intensities of each cell line relative to controls, defined as 1.0. (B) The same batch of cell lysates was subjected to apoptosis ELISA. Data show a representative of three independent experiments. Bars, SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Knockdown, Expressing, Infection, Control, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin inhibited tumor growth and induced apoptosis and TRAIL expression in a tumor xenograft model (A) Tumor growth curves were plotted using average tumor volume within each group at the indicated time points. A two-tailed Student’s t test was used for statistical analysis (∗p < 0.01, ∗∗p < 0.003). Bars: SD. (B and C) At the end of treatment, tumor-bearing mice from control group and metformin-treated group were sacrificed. The tumors were dissected, imaged as indicated (B), and measured for weight (C). (D and E) Cell lysis from the tumor tissues was prepared. Western blot assays were performed to examine the expression of TRAIL, DR4, DR5, and apoptotic markers PARP, caspase-8, and caspase-3. β-actin was used as an internal control. The densitometry analyses of TRAIL signals are shown underneath, and the arbitrary numbers indicate the intensities of each tumor relative to control 1, defined as 1.0. (F) Formalin-fixed paraffin-embedded sections of xenograft tumors were analyzed with H&E staining, IHC staining for Ki67, TRAIL, and cleaved caspase-3. Scale bar, 210 μm. Quantification of IHC staining with ImageJ and ImageJ plugin IHC profiler are shown underneath.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Expressing, Two Tailed Test, Control, Lysis, Western Blot, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry

Journal: Journal of cellular biochemistry

Article Title: Targeting the epithelial-mesenchymal transition (EMT) pathway with combination of Wnt inhibitor and chalcone complexes in lung cancer cells.

doi: 10.1002/jcb.30442

Figure Lengend Snippet: FIGURE 7 Determination of cleaved caspase 3/7 and cleaved PARP levels in A549 (A, B) and H1299 (C, D) cells after treatment with the Complex 1, Complex 2 (6.25 µM), and their Niclosamide (Niclo) (1.25 µM) combination for 48 h by ELISA assay. Denotes statistically significant differences between complex 1 or 2 and their combination with niclosamide: (*p < 0.05); (**p < 0.01); (***p < 0.001) and between niclosamide and their combination with complexes 1 or 2 (#p < 0.05); (##p < 0.01); (###p < 0.001). Data are presented as mean ± SD (n = 3). ELISA, enzyme‐linked immunosorbent assay; SD, standard deviation.

Article Snippet: After treatment, cleaved PARP levels were calculated using the PARP (Cleaved) [214/215] Human ELISA kit (Cellsignalling, #7262) according to the protocol described in the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: MEIS and PARP1 interact. (A) Mass spectrometry scores of PARP1 and PARP1-interacting proteins copurifying with MEIS2 from nuclear extracts of SK-N-BE(2) neuroblastoma cells. (B and C) PARP1 (B) and MEIS2 (C) protein distribution in the RMS. MEIS2 and PARP1 protein staining is shown in brown, and nuclear counterstaining is in blue. CC, corpus callosum. (D) Domain structure of PARP1. (E) GST pulldown of HA-tagged MEIS2 with different PARP1–GST fusion proteins. The left panel shows a blot probed for HA detecting MEIS2-HA, and the right panel shows the same blot probed for GST detecting the different PARP1–GST fusion proteins. Because transfer of full-length PARP1–GST was incomplete because of its large size, higher-exposure images of the blot probed for HA and GST are shown in Fig. S2. The number of biological replicates performed for E are listed in Table S4. PD, pulldown; WB, Western blot.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Mass Spectrometry, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: MEIS recruits PARP1 to the Dcx promoter/enhancer. (A) ChIP-qPCR for PARP1 at Dcx(−2.7) during neuronal differentiation. (B) Comparison of PARP1 binding to Dcx(−2.7) and “ primers out ” (−12) at 0 and 5 h; values for Dcx(−2.7) correspond with those shown in A. (C) ChIP-qPCR for H1 at Dcx(−2.7) and “ primers out ” at the times indicated. (D) ChIP-qPCR for PARP1 and H1 at the Myog promoter, showing reciprocal binding of PARP1 and H1. (E and F) ChIP-reChIP for H1 followed by PAR at Dcx(−2.7) (E) and “ primers out ” (F) at 3 h of differentiation. (G) In vitro PARylation assay of recombinant PARP1 and H1 in the presence of biotinylated (biot.) NAD + and stimulated by the addition of low-molecular DNA fragments (sheared DNA), demonstrating efficient PARylation of H1 and autoPARylation of PARP1. The asterisks in A–F indicate statistically significant enrichment of ChIP with the antibodies indicated relative to ChIP with the IgG control antibodies for the same conditions, with *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical significance of ChIP results between experimental groups is given as p = numerical value. ChIP data are represented as means ± SEM. Samples sizes and the number of biological replicates are listed in Table S4. IP, immunoprecipitation; WB, Western blot.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Binding Assay, In Vitro, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: MEIS recruits PARP1 to Dcx(−2.7) . (A and B) ChIP-qPCR for the proteins indicated at 5 h of differentiation in cells transfected with Meis1/2 -specific siRNAs (gray bars) or nontargeting control siRNAs (black bars): MEIS, PARP1, and PBX1 at Dcx(−2.7) (A), and H1 at Dcx(−2.7) (B) . (C) ChIP-qPCR for H1 at Dcx(−2.7) in undifferentiated cell cultures. H1 is not released from the Dcx promoter/enhancer when cells are subjected to the cellular differentiation protocol under Meis -knockdown conditions. The asterisks indicate statistically significant enrichment of ChIP with the antibodies indicated relative to ChIP with the IgG control antibodies for the same conditions, with *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical significance of ChIP results between experimental groups is given as p = numerical value. ChIP data are represented as means ± SEM. Samples sizes and the number of biological replicates are listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Transfection, Cell Differentiation

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: PARP activity is required for neuronal differentiation and H1 eviction from the Dcx promoter/enhancer. (A) Schematic outline of the experiments shown in B–F. (B–F) Reduced neurogenesis and enhanced astrogliogenesis upon pharmacological PARP inhibition: (B and C) Proportions of neurons (B) and astrocytes (C) generated in the presence of increasing concentrations of Olaparib. (D) Representative images of cultures differentiated in the presence of Olaparib or 0.01% DMSO as control. Arrowheads indicate DCX-positive neuronal processes. (E and F) Neurons and astrocytes generated in the presence of 3AB (E) or PJ34 (F). (G, left) Outline of the experiment; (right) neuronal differentiation after shRNA-mediated knockdown of PARP1. (H, left) Outline of the experiment; (right) qPCR for Meis2 , Pbx1 , and Dcx transcripts in cells differentiated for 10 h by growth factor removal and plating on laminin in the presence of 100 nM Olaparib. Expression is shown relative to expression determined in cells treated with 0.01% DMSO (vehicle only). (I, left) Outline of the experiment; (right) ChIP-qPCR for H1 at Dcx(−2.7) in cells differentiated for 5 h in the presence of 6 mM 3AB (gray bars) or water as vehicle control (black bars). Statistical significance of ChIP results between experimental groups is given as p = numerical value. Data are represented as means ± SEM. Samples sizes and the number of biological replicates are listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Activity Assay, Inhibition, Generated, shRNA, Expressing

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: Neuronal differentiation is not compromised when pharmacological inhibition of PARP follows the induction of cellular differentiation. (A, left) Outline of the experiment: differentiation was induced in primary SVZ progenitor cells by growth factor removal and plating on laminin 12 h before addition of PJ34 to the culture medium. Addition of water (diluent) served as control; (right) proportion of TuJ1 + neurons generated under both conditions; after 3 d of differentiation, 53.9 ± 3.6% (SD) of the cells differentiated into DCX + neurons under standard conditions, and 49.9 ± 7.6% (SD) differentiated when PJ34 was added to the medium after the first 12 h of differentiation. (B) Representative micrographs of these experiments. Data are represented as means ± SEM, and the number of biological replicates is listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Inhibition, Cell Differentiation, Generated

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: Identification of PARP-regulated genes by Affymetrix Mouse Gene 1.0 ST arrays. (A) Scatter blot of differentially regulated genes in adult SVZ progenitor cells differentiated in the presence or absence of 100 nM Olaparib; representative significantly up-regulated genes are highlighted in red, and representative significantly down-regulated genes are in green. A list of all significantly differentially expressed genes is given in Table S5. (B) GO term enrichment analysis for genes differentially expressed after Olaparib treatment relative to the control. A more detailed collection of GO terms is shown in Table S1. (C) Transcript expression of six down-regulated candidate genes in SVZ-derived stem and progenitor cells differentiated for 10 h in vitro upon pharmacological inhibition of PARP1 and PARP2 or Meis1/2 knockdown, respectively, validated by qPCR. Gray bars, PARP-inhibition: transfection with nontargeting siRNAs and treatment with 100 nM Olaparib; black bars, Meis knockdown: transfection with Meis1/2 -specific siRNAs and treatment with 0.01% DMSO. Transcript levels are expressed relative to those determined under control conditions (transfection with nontargeting siRNAs and differentiation in the presence of 0.01% DMSO). (D) ChIP-qPCR for MEIS2 and PARP1 at a consensus-binding motif for MEIS/PBX-type HD proteins upstream of the Draxin transcriptional start site in adult SVZ progenitor cells after 5 h of neuronally directed differentiation. Gray bars, cells transfected with Meis1/2 -specific siRNAs; black bars, cells transfected with nontargeting control siRNAs. (E) ChIP-qPCR for MEIS2 and PARP1 under identical conditions as shown in D but binding to a consensus motif for MEIS/PBX-type HD proteins upstream of the Nrep transcriptional start site was assessed. (F–I) Transcript expression for Draxin (F and G) and Nrep (H and I) in the adult SVZ visualized by in situ hybridization in comparison with MEIS2 and DCX protein expression. (F and H) MEIS2 (red) and DCX (green) cell nuclei are counterstained with DAPI (blue). (G and H) Overlay of transcript expression (purple) and MEIS2 protein (red). The number of biological replicates is listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Expressing, Derivative Assay, In Vitro, Inhibition, Transfection, Binding Assay, In Situ Hybridization

Journal: Advanced Science

Article Title: Nanoparticles Dysregulate the Human Placental Secretome with Consequences on Angiogenesis and Vascularization

doi: 10.1002/advs.202401060

Figure Lengend Snippet: Study workflow. a) Schematic representation of the placental structure at first trimester and term. b) Experimental strategy showing the main steps of the study. H&E: hematoxylin‐eosin; hNPC: human neural progenitor cells; HUVEC: human umbilical cord endothelial cells; NP: nanoparticles; trim: trimester; PARP: poly(ADP‐ribosyl) polymerase.

Article Snippet: Cell death was determined by quantification of PARP cleavage in whole tissue lysates, prepared from first trimester and term explants, using PARP (Cleaved) [214/215] human ELISA Kit (KHO0741, Thermofisher) and following manufacturer's instructions.

Techniques:

Journal: Advanced Science

Article Title: Nanoparticles Dysregulate the Human Placental Secretome with Consequences on Angiogenesis and Vascularization

doi: 10.1002/advs.202401060

Figure Lengend Snippet: Effect of TiO 2 , SiO 2 NPs, and DEPs on human placental tissue integrity, viability, and functionality after 48 h of exposure. Explants from first trimester (a) and term (b) placentae were exposed to TiO 2 , SiO 2 NPs, (1 and 25 µg mL −1 ) or DEPs (0.45 µg mL −1 ) and characterized for histological and immunohistochemical changes by staining for H&E and trophoblast marker CK‐7, respectively. Representative images from three independent biological experiments are shown. Scale bars = 50 µm. c) PARP cleavage after 48 h of particle exposure in the tissue explants. Data represent the mean (± SEM) fold changes (FC) compared to the untreated control of n = 3–9 biologically independent samples (First trim: untreated n = 7, all others n = 4; Term: untreated n = 9, Staurosporine (positive control) n = 3, all others n = 5) d) Effect of NPs on hCG release from first trimester and term placental explants. Data represent the mean (± SEM) FC compared to the untreated control of n = 4–8 biologically independent samples (First trim: untreated n = 8, all others n = 4; Term: untreated n = 8, DEPs n = 5, all others n = 4). One‐way ANOVA with Dunnett's multiple comparisons correction was used for the analysis of comparisons between control (untreated) and the treatments (c) * p < 0.0001. CK‐7: cytokeratin‐7; hCG: human chrionic gonadotropin; PARP: poly(ADP‐ribosyl) polymerase.

Article Snippet: Cell death was determined by quantification of PARP cleavage in whole tissue lysates, prepared from first trimester and term explants, using PARP (Cleaved) [214/215] human ELISA Kit (KHO0741, Thermofisher) and following manufacturer's instructions.

Techniques: Immunohistochemical staining, Staining, Marker, Control, Positive Control