Journal: Molecular human reproduction

Article Title: Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

doi: 10.1093/molehr/gaw065

Figure Lengend Snippet: Figure 2 Effect of S1P on the expression of endothelial adherens junction proteins in endothelial cells in the presence of FF from controls and patients at risk of OHSS. Densitometric quantification of (A) phospho-vascular endothelial (p-VE)-cadherin, (B) VE-cadherin, (C) N-cadherin and (D) β-catenin. Representative immunoblots are shown in the lower panel. Data are expressed as means ± SEM normalized to β-actin of three independent experiments using 20 control patients and 20 OHSS patients at risk of OHSS (*P < 0.05).

Article Snippet: The blot was preincubated in blocking buffer (5% nonfat milk, 0.05% Tween 20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated overnight in blocking buffer at 4°C with appropriate primary antibodies: β-actin 1/3000 (sc-1616-R), N-cadherin 1/200 (sc-7939), vascular endothelial (VE)-cadherin 1/100 (sc-9989) and β-catenin 1/200 (sc-59737) (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), phospho-VE-cadherin 1/1000 (MAB1002) (Millipore, MA, USA), VEGF 1/1000 (ab46154) (Abcam, Cambridge, USA), KDR (VEGFR-2) 1/1000 (D5B1) (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: iScience

Article Title: Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells

doi: 10.1016/j.isci.2021.103605

Figure Lengend Snippet:

Article Snippet: Alexa Fluor® 488 Anti-BrdU antibody [BU1/75 (ICR1)] , Abcam , #ab220074.

Techniques: Produced, Recombinant, Enzyme-linked Immunosorbent Assay, Adhesive, Western Blot, CRISPR, Control, Plasmid Preparation, Software

Journal: Acta Pharmacologica Sinica

Article Title: TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I

doi: 10.1038/s41401-023-01091-w

Figure Lengend Snippet: a Complex I activity assay showed that 450 nmol/L DPT enhanced complex I activity in oxidation of NADH in a time-dependent manner. b Representative images of confocal microscopy combined with immunochemical staining showed ND1 level was increased obviously in the mitochondria of the cells incubated with 450 nmol/L DPT for 24 h. c Western blotting analysis revealed 450 nmol/L DPT upregulated both ND1 and ND2 in a time-dependent manner. d Co-immunoprecipitation analysis showed that the protein levels of ND2, NDUFS2 and NDUFS4 co-immunoprecipitated with ND1 were increased by 450 nmol/L DPT at 12 h, which became more apparent when incubation time of DPT was increased to 24 h. e Complex I activity assay showed that the increased complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h was inhibited by pretreatment with 2 μmol/L rotenone for 1 h. f Western blotting revealed that knockdown of ND1 with siRNA not only apparently inhibited ND1 upregulation in mitochondria, but also alleviated nuclear translocation of AIF induced by 450 nmol/L DPT at 24 h. g Complex I activity assay showed that the increased complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h was inhibited in the cells transfected with ND1 siRNA. h Statistical analysis of the intensity of the red fluorescence detected by MitoSOX red revealed that the increased mitochondrial superoxide induced by 450 nmol/L DPT at 24 h was suppressed when ND1 was knocked down with siRNA. i Complex I activity assay showed that knockdown of ND1 with siRNA significantly prevented the increase of complex I activity in oxidation of NADH caused by 450 nmol/L DPT at 24 h. j Western blotting revealed knockdown of TAX1BP1 with siRNA prevented the upregulation of ND1 and ND2 and downregulation of GPX4 and catalase in mitochondrial fractions induced by 450 nmol/L DPT at 24 h. k Co-immunoprecipitation analysis showed that the enhanced interactions between ND1, ND2, NDUFS2 and NDUFS4 caused by 450 nmol/L DPT at 24 h were all inhibited when TAX1BP1 was knocked down with siRNA. * P < 0.01 versus control group. The values are expressed as mean ± SD ( n = 5 per group).

Article Snippet: The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA).

Techniques: Activity Assay, Confocal Microscopy, Staining, Incubation, Western Blot, Immunoprecipitation, Knockdown, Translocation Assay, Transfection, Fluorescence, Control

Journal: Acta Pharmacologica Sinica

Article Title: TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I

doi: 10.1038/s41401-023-01091-w

Figure Lengend Snippet: a Representative images of nude mice with xenografted gliomas showed that tumor growth was significantly inhibited when the mice were treated with DPT at a dose of 10 mg/kg for 8 consecutive days. b Statistical analysis of the tumor volumes confirmed that the growth of the tumor in vivo was significantly inhibited by DPT. c Western blotting analysis revealed that DPT promoted mitochondrial translocation of TAX1BP1 and nuclear translocation of AIF. It also induced marked upregulation of ND1 and ND2 and downregulation of mitochondrial GPX4 and catalase in mitochondrial fractions, and obvious upregulation of PARP1, PAR, p-ATM and p-H2AX in nuclear fractions. d NAD + assay showed that NAD + was decreased significantly in DPT-treated group when compared with that in control group. e Complex I activity assay showed that DPT treatment resulted in upregulation of complex I activity in oxidation of NADH in vivo. f H 2 O 2 assay proved the level of H 2 O 2 was increased obviously by DPT in vivo. g Immunoprecipitation with an antibody against ND1 revealed that the co-immunoprecipitated ND2, NDUFS2 and NDUFS4 with ND1 were all significantly increased in the DPT-treated group. The values are expressed as mean ± SD ( n = 6 per group).

Article Snippet: The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA).

Techniques: In Vivo, Western Blot, Translocation Assay, Control, Activity Assay, Immunoprecipitation

Journal: Acta Pharmacologica Sinica

Article Title: TAX1BP1 contributes to deoxypodophyllotoxin-induced glioma cell parthanatos via inducing nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I

doi: 10.1038/s41401-023-01091-w

Figure Lengend Snippet: DPT induces PARP1 over-activation via causing DNA double strand breaks (DSBs). The activated PARP1 promotes TAX1BP1 translocation from cytoplasm to mitochondria by depletion of its substrate NAD + . Then, TAX1BP1 enhances the activation of respiratory chain complex I not only by increasing the protein levels of ND1 and ND2, but also via promoting their assemblies into complex I. The activated respiratory chain complex I generates more superoxide, resulting AIF translocation from mitochondria to nuclei. Within nuclei, AIF serves as a nuclease to cause chromatinolysis to complete the final stage of parthanatos.

Article Snippet: The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA).

Techniques: Activation Assay, Translocation Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: A QPCR results showed that CB1 was downregulated in colorectal cancer cells. B QPCR results showed that EGFR was upregulated in colorectal cancer cells. C, D Western blot analysis showed that CB1 was downregulated and EGFR was upregulated in colorectal cancer cells. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: A Western blot assay tested the protein level of CB1 after ACEA and AM251 treatment. B Western blot analysis showed that CB1 activation significantly decreased the expression of EGFR while its inhibition increased the expression of EGFR. C, D Colony forming assay showed that CB1 activation suppressed colony formation of colorectal cancer cells, while its inhibition promoted colony formation. E, F Wound healing test showed that CB1 activation prevented cell migration while CB1 inhibition enhanced cell migration of colorectal cancer cells. G, H Transwell invasion assay demonstrated that CB1 activation blocked cell invasion while CB1 inhibition enhanced cell invasion of colorectal cancer cells. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Migration, Transwell Invasion Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Cells were transfected with EGFR plasmids and simultaneously treated with ACEA. A QPCR analysis showed that EGFR plasmids transfection increased the expression of EGFR in SW480 and SW620 cells. B , C Colony-forming assay demonstrated that EGFR overexpression blocked the proliferation suppression induced by CB1 activation. D , E Wound healing test showed that EGFR overexpression counteracted the migration inhibition induced by CB1 activation. F , G Transwell invasion assay showed that EGFR overexpression prevented the decrease in cell invasion caused by CB1 activation. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, Expressing, Over Expression, Activation Assay, Migration, Inhibition, Transwell Invasion Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Colorectal cancer cells were transfected with EGFR plasmids and simultaneously treated with ACEA. Then PMA induced THP-1 cells were co-cultured with the culture medium of colorectal cancer cells. A – F QPCR results demonstrated that EGFR overexpression blocked CB1 activation caused increase in the expression of IL-6 and TNF-α, and decrease in IL-10, CCL22, Arg-1 and CD206. G – J ELISA assay showed that EGFR overexpression blocked CB1 activation caused increase in the release of IL-6 and TNF-α, and decrease in the release of IL-10 and CCL22. K , L Western blot showed that EGFR overexpression prevented the decrease in the expression of Arg-1 and CD206. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, Cell Culture, Over Expression, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Colorectal cells were transfected with plasmids bearing shRNA against EGFR and simultaneously treated with AM251. Culture medium for these colorectal cells were taken to incubate PMA-induced THP-1 cells. A QPCR analysis showed that sh-EGFR effectively suppressed the expression of EGFR. B , C Colony formation test demonstrated that EGFR knockdown reversed the enhancement in colony formation induced by AM251. D – I QPCR results demonstrated that EGFR knockdown reversed the CB1 inhibition caused decrease in the expression of IL-6 and TNF-α, and increase in IL-10, CCL22, Arg-1 and CD206. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, shRNA, Expressing, Knockdown, Inhibition

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: SW480 cells were subcutaneously injected into nude mice and the tumors were treated with control or ACEA (1.5 mg/kg/d). A CB1 activation suppressed tumor growth. Representative tumors ( B ) and tumor weight ( C ) from different experimental groups. D QPCR analysis showed that CB1 activation increased the expression of IL-6 and TNF-α, and decreased the expression of IL-10, CCL-22, Arg-1 and CD206. E Western blot analysis showed that the expression of EGFR, Arg-1 and CD206 were decreased in the tumors of ACEA-treated mice. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Injection, Control, Activation Assay, Expressing, Western Blot

Journal: The FASEB Journal

Article Title: G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR

doi: 10.1096/fj.201900233rr

Figure Lengend Snippet: Figure 2. Down-regulation of G9a represses cell proliferation of GC. A) Western blotting and real-time qPCR assays were performed to detect the G9a expression in 3 G9a-knockdown GC cell lines. Tubulin was used as a loading control. B) G9a knockdown repressed the growth and proliferation of HGC-27, MKN-45, and SGC-7901 cells. Cell viability was detected using CCK8 assays. C) GC cells were treated with BIX01294 and analyzed for cell viability using CCK8 assays. D) Ki67 immunofluorescence assays were performed after G9a knockdown or inhibition. Cells were treated with 7 mM BIX01294 for 48 h. Representative images show immunofluorescence. Scale bars, 20 mm. All data are shown as means 6 SD; n = 3. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: G9a (ab40542), Ki67 (ab92742), Unc-51–like autophagyactivating kinase 1 (ULK1; ab128859), Beclin-1 (ab207612), p62/ SQSTM1 (ab207305), mTOR (ab32028), H3K9me1 (ab9045), H3K9me2 (ab1220), and p-H3S10 (5176) antibodies were purchased from Abcam (Cambridge, MA, USA). p(Ser2448)-mTOR (D9C2), CyclinA2 (BF683), CyclinB1 (D5C10), p(Ser757)-ULK1 (D7O6U), p70S6K (D5U1O), CDK1 (POH1), and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Knockdown, Control, Inhibition

Journal: The FASEB Journal

Article Title: G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR

doi: 10.1096/fj.201900233rr

Figure Lengend Snippet: Figure 3. Down-regulation of G9a represses colony formation in vitro and tumor formation of GC cells in vivo. A, B) The effects of G9a on the colony formation in 3 G9a-knockdown GC cell lines. Scale bars, 20 mm. The colony numbers in plate were quantified. C, D) The tumor growth curve and tumor weight of G9a knockdown GC cells injected into NOD-SCID mice. E) Immunohistochemical (IHC) staining of G9a expression (left) and Ki67 expression (right). Scale bars, 20 mm. F) The quantification of G9a-positive cells (left) and Ki67-positive cells (right). All data are shown as means 6 SD; n = 3. **P , 0.01, ***P , 0.001.

Article Snippet: G9a (ab40542), Ki67 (ab92742), Unc-51–like autophagyactivating kinase 1 (ULK1; ab128859), Beclin-1 (ab207612), p62/ SQSTM1 (ab207305), mTOR (ab32028), H3K9me1 (ab9045), H3K9me2 (ab1220), and p-H3S10 (5176) antibodies were purchased from Abcam (Cambridge, MA, USA). p(Ser2448)-mTOR (D9C2), CyclinA2 (BF683), CyclinB1 (D5C10), p(Ser757)-ULK1 (D7O6U), p70S6K (D5U1O), CDK1 (POH1), and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, In Vivo, Knockdown, Injection, Immunohistochemical staining, Immunohistochemistry, Expressing