Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Protective Effect of XinJiaCongRongTuSiZiWan on the Reproductive Toxicity of Female Rats Induced by Triptolide

doi: 10.1155/2022/3642349

Figure Lengend Snippet: XJCRTSZW ameliorates TP-induced reproductive toxicity via autophagy by inhibiting the AMPK α -1/SIRT1/Akt signaling axis. (a, b) The expression level of AMPK, SIRT1, and 8-OhdG in GCs was detected using IHC. (c) The autophagosome in GCs was evaluated using a transmission electron microscope. (d–g) The protein levels of beclin-1, LC3-II/LC3-I, cleaved-caspase-3, p62, procaspase-3, p-AMPK α -1, p-SIRT1, p-Akt, AMPK α -1, SIRT1, and Akt were determined using the Western blot. The data were expressed after being normalized to β -actin. The means ± SD of five independent samples were shown. ∗ p < 0.05 compared to the control group. # p < 0.05 compared to the TP group. & p < 0.05 compared to the TP + XJCRTSZW-high group.

Article Snippet: Sections were stained with rabbit polyclonal SIRT1 antibody (1 : 100, bs-5973R, Bioss, Beijing, China), rat monoclonal AMPK antibodies (1 : 100, 66536-1-Ig, Bioss), and rabbit polyclonal 8-OHdG antibody (1 : 100, bs-1278R, Bioss) overnight at 4°C.

Techniques: Expressing, Transmission Assay, Microscopy, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Protective Effect of XinJiaCongRongTuSiZiWan on the Reproductive Toxicity of Female Rats Induced by Triptolide

doi: 10.1155/2022/3642349

Figure Lengend Snippet: TP-induced autophagy of human ovarian granulosa cells through the AMPK α -1/SIRT1/Akt signaling axis. (a) The autophagosome in human ovarian granulosa cells was evaluated using transmission electron microscope. (b, c) The protein level of beclin-1, LC3-II/LC3-I, cleaved-caspase-3, p62, procaspase-3, p-AMPK α -1, p-SIRT1, p-Akt, AMPK α -1, SIRT1, and Akt was determined using the Western blot. The data were expressed after being normalized to β -actin. The means ± SD of three independent samples were shown. ∗ p < 0.05 compared to the control group. # p < 0.05 compared to the TP group. & p < 0.05 compared to the TP + XJCRTSZW group.

Article Snippet: Sections were stained with rabbit polyclonal SIRT1 antibody (1 : 100, bs-5973R, Bioss, Beijing, China), rat monoclonal AMPK antibodies (1 : 100, 66536-1-Ig, Bioss), and rabbit polyclonal 8-OHdG antibody (1 : 100, bs-1278R, Bioss) overnight at 4°C.

Techniques: Transmission Assay, Microscopy, Western Blot

Journal: Nucleic Acids Research

Article Title: Dicer interacts with SIRT7 and regulates H3K18 deacetylation in response to DNA damaging agents

doi: 10.1093/nar/gkv1504

Figure Lengend Snippet: Direct interaction between Dicer and SIRT7. ( A ) Co-IP of endogenous Dicer and SIRT7 in HEK293T and HCT116 cells using anti-Dicer antibody. ( B ) Co-IP of endogenous Dicer and SIRT7 using two different anti-SIRT7 antibodies (H00051547-D01 and 5360). ( C ) Anti-SIRT7 (5360) co-IP using HCT116 cell lysate in the presence of increasing NaCl concentrations. ( D ) Anti-SIRT7 (5360) co-IP using HCT116 cell lysate in the presence or absence of RNases. ( E ) Direct interaction of Dicer and SIRT7 revealed by co-IP using purified recombinant Dicer and SIRT7 proteins. The recombinant proteins and IP antibodies added in each reaction are indicated on the top. BSA was used to compensate the missing protein when only one protein (Dicer or SIRT7) was included in the assay. ( F ) Interaction between recombinant human Dicer protein and purified His-tagged SIRT7 protein revealed by in vitro binding assay. The proteins loaded to the His-tag purification column are indicated on the top. B, representative bound fraction, UB, representative unbound fraction. ( G ) Anti-FLAG M2 gel pulled down endogenous Dicer in extracts of stable Flag-SIRT7(WT)- or Flag-SIRT7 (S111A)-expressing HEK293T cells, but not in extracts of Flag-SIRT7(dE2)-expressing HEK293T cells. Ctr: HEK293T cells transfected with the empty vector pcDNA3.1.

Article Snippet: Three hours later, the reaction mixture was added with anti-SIRT7 antibody (H00051547-B01, Abnova), anti-Dicer antibody, or IgG control, and continued to incubate at 4°C overnight before precipitation with Protein G Sepharose beads.

Techniques: Co-Immunoprecipitation Assay, Purification, Recombinant, In Vitro, Binding Assay, Expressing, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Dicer interacts with SIRT7 and regulates H3K18 deacetylation in response to DNA damaging agents

doi: 10.1093/nar/gkv1504

Figure Lengend Snippet: Colocalization of Dicer and SIRT7 in the cytoplasm. ( A ) Subcellular localization of Dicer, SIRT7, H3K18Ac, histone H3 and lamin A/C, revealed by biochemical fractionation. Upper panel, the schematic diagram of biochemical fractionation assay; bottom panel, the representative western blot images. S2, S3 and S4 represent the cytoplasmic, the nucleoplasmic and the chromatin-associated fractions, respectively. ( B and C ) Colocalization of Dicer and SIRT7 in the cytoplasm revealed by immunofluorescence using anti-Dicer and two different anti-SIRT7 antibodies. (B) NB110–81762; (C) 5360.

Article Snippet: Three hours later, the reaction mixture was added with anti-SIRT7 antibody (H00051547-B01, Abnova), anti-Dicer antibody, or IgG control, and continued to incubate at 4°C overnight before precipitation with Protein G Sepharose beads.

Techniques: Fractionation, Western Blot, Immunofluorescence

Journal: Nucleic Acids Research

Article Title: Dicer interacts with SIRT7 and regulates H3K18 deacetylation in response to DNA damaging agents

doi: 10.1093/nar/gkv1504

Figure Lengend Snippet: Dicer expression level affects SIRT7 subcellular distribution and H3K18Ac level in HEK293T cells. ( A and B ) Co-IP of Dicer and SIRT7 using excessive amount of anti-Dicer antibody in pDESTmycDICER (A) or siDicer1 (B) transiently transfected cells. ( C ) Increased level of chromatin-associated SIRT7 and decreased level of cytoplasmic SIRT7 in siDicer-transfected cells, revealed by biochemical fractionation. ( D ) Decreased level of chromatin-associated SIRT7 and increased level of cytoplasmic SIRT7 in pDESTmycDICER transiently transfected cells, revealed by biochemical fractionation. ( E and F ) Representative western blot images of Dicer, H3K18Ac, SIRT7 and histone H3 in pDESTmycDICER (E) or siDicer (F) transiently transfected cells. S2, S3 and S4 represent the cytoplasmic, the nucleoplasmic and the chromatin-associated fractions, respectively.

Article Snippet: Three hours later, the reaction mixture was added with anti-SIRT7 antibody (H00051547-B01, Abnova), anti-Dicer antibody, or IgG control, and continued to incubate at 4°C overnight before precipitation with Protein G Sepharose beads.

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Fractionation, Western Blot

Journal: Nucleic Acids Research

Article Title: Dicer interacts with SIRT7 and regulates H3K18 deacetylation in response to DNA damaging agents

doi: 10.1093/nar/gkv1504

Figure Lengend Snippet: DNA damaging agents upregulate Dicer, causing a decrease of chromatin-associated SIRT7 and an increase of H3K18Ac in HEK293T cells. ( A ) Representative western blot images of Dicer, H3K18Ac, SIRT7 and histone H3 in cells treated with different doses of DNA damaging agents. ( B ) Co-IP of Dicer and SIRT7 using excessive amount of anti-Dicer antibody in DNA damaging treated cells. ( C and D ) Decreased level of chromatin-associated SIRT7 and increased level of cytoplasmic SIRT7 in DDP- or doxo-treated cells (C) or IR-treated cells (D), revealed by biochemical fractionation. S2, S3 and S4 represent the cytoplasmic, the nucleoplasmic and the chromatin-associated fractions, respectively.

Article Snippet: Three hours later, the reaction mixture was added with anti-SIRT7 antibody (H00051547-B01, Abnova), anti-Dicer antibody, or IgG control, and continued to incubate at 4°C overnight before precipitation with Protein G Sepharose beads.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Fractionation

Journal: Nucleic Acids Research

Article Title: Dicer interacts with SIRT7 and regulates H3K18 deacetylation in response to DNA damaging agents

doi: 10.1093/nar/gkv1504

Figure Lengend Snippet: Dicer knockdown prevents the reduction of chromatin-associated SIRT7 and the increase of H3K18Ac in DNA damaging treated HEK293T cells. ( A ) Dicer knockdown blocked the increase of SIRT7 in the cytoplasmic fraction of DNA damaging treated cells. ( B ) Dicer knockdown prevented the reduction of SIRT7 in the chromatin-associated fraction of DNA damaging treated cells. ( C ) Dicer knockdown partially prevented the increase of H3K18Ac in DNA damaging treated HEK293T cells. ( D ) Quantification of H3K18Ac levels in (C). Fold induction of H3K18Ac by DNA damaging agents = the level of H3K18Ac in DNA damaging treated cells/the level of H3K18Ac in non-DNA damaging treated cells. * P < 0.05.

Article Snippet: Three hours later, the reaction mixture was added with anti-SIRT7 antibody (H00051547-B01, Abnova), anti-Dicer antibody, or IgG control, and continued to incubate at 4°C overnight before precipitation with Protein G Sepharose beads.

Techniques: Knockdown

Journal: Oncotarget

Article Title: Sirtuin 7: a new marker of aggressiveness in prostate cancer

doi: 10.18632/oncotarget.20468

Figure Lengend Snippet: (A) Sirtuin 7 immunohistochemical staining on human prostate tissue. (A.a) Healthy prostate, absence of staining. (A.b) Prostate adenocarcinoma, nuclear staining of tumor cells. (A.c) On the edge of the tumor, no staining of healthy glands, nuclear staining of tumor cells. (A.d) Nucleolar staining of anti-SIRT7 antibody within a tumor area (arrow). (B) Association between All red Score and Gleason Score of index lesion in each surgical specimen. (C) Analysis of index lesion All red Score distribution according to the absence or the presence of prostatic capsular invasion in each surgical specimen. (D) Analysis of SIRT7 and AMACR mRNA expression in prostatic tumor cells.

Article Snippet: Immunoblotting was performed with antibodies against SIRT7 (Merck-Millipore, Molsheim, France), HSP90 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Acetyl Histone H3K18 (Euromedex, France).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: Sirtuin 7: a new marker of aggressiveness in prostate cancer

doi: 10.18632/oncotarget.20468

Figure Lengend Snippet: (A) Western blot analysis of SIRT7 and acetylated H3K18 in LNCaP (1), P69 (2), 22RV (3), DU145 (4) and PC3 (5) cells. (B) Western blot was performed to analyze SIRT7 expression in DU145 and PC3 cells transfected with siRNA. (C) Cell viability of DU145 and PC3 cells transfected with control SiRNA (SiCT) and SIRT7 siRNA (siSIRT7). The data represents three independent experiments with sem (D) Migration assay: the graph represents the number of cells/mm 2 migrating across the boyden chamber in three independent experiments performed in triplicate as described in the materials and methods. (Student t-Test ** p <0.01).

Article Snippet: Immunoblotting was performed with antibodies against SIRT7 (Merck-Millipore, Molsheim, France), HSP90 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Acetyl Histone H3K18 (Euromedex, France).

Techniques: Western Blot, Expressing, Transfection, Control, Migration

Journal: Oncotarget

Article Title: Sirtuin 7: a new marker of aggressiveness in prostate cancer

doi: 10.18632/oncotarget.20468

Figure Lengend Snippet: (A) Western blot representing the expression of SIRT7 in the different clones: Control cells (CT), cells expressing SIRT7wt and SIRT7mut. (B) The graphs represent the number of cell/mm2 migrating across the boyden chamber in a migration and invasion assay. Three independent experiments were performed in triplicate as described in the materials and methods. (C) q-PCR of the expression of fibronectin, Slug and vimentin in the different populations of cells. The graph represents the results of five independent experiments. (D) Western blot showing the expression of fibronectin, vimentin, Slug, SIRT7 and HSP90 in SIRT7wt and SIRT7mut. (E) Viability assay in the different clones treated for 72h with the indicated concentrations of docetaxel. The graph represents the mean of four independent experiments performed in quadruplicate. (Student t-Test; * p <0.05; ** p <0.01; *** p <0.001).

Article Snippet: Immunoblotting was performed with antibodies against SIRT7 (Merck-Millipore, Molsheim, France), HSP90 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Acetyl Histone H3K18 (Euromedex, France).

Techniques: Western Blot, Expressing, Clone Assay, Control, Migration, Invasion Assay, Viability Assay