Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Modulation of fatty-acid-synthesis related genes by globular adiponectin in breast cancer cells. MCF-7 (A, C, E, and H) and MDA-MB-231 (B, D, F, G, and I) cells were treated with gAcrp (1 μg/mL) for indicated time periods. A-B The expression levels of precursor and nuclear SREBP-1 were determined by western blot analysis. C-D SREBP-1 mRNA levels were measured by RT-qPCR. E-F The SREBP-1 specific dsDNA binding activity was examined as described in Methods. G The expression of the genes modulating fatty acid synthesis, including FASN, ACC-1, ACLY, FADS1, FADS2, and SCD-1, were analyzed by RT-qPCR. H-I The protein levels of FASN were examined by western blot analysis. * indicates p < 0.05 compared to control; n=3 except where specifically indicated in Figures

Article Snippet: The specific transcription factor DNA binding activity was examined using the SREBP-1 Transcription Factor Assay Kit from Abcam (#ab133125) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Binding Assay, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Roles of SIRT-1 in fatty acid metabolic reprogramming by globular adiponectin in breast cancer cells. A-B MCF-7 ( A ) and MDA-MB-231 ( B ) cells were treated with gAcrp as indicated. SIRT-1 expression was determined by western blot analysis. C MCF-7 cells were pretreated with EX527 (5 μM) for 2 h, followed by incubation with gAcrp (1 μg/mL) for further 8 h. The protein levels of SREBP-1 and FASN were examined by western blot analysis. D MCF-7 cells were transfected with a siRNA targeting SIRT-1 or a scramble control siRNA for 36 h, followed by treatment with gAcrp (1 μg/mL) for 8 h. (Upper panel) The gene silencing efficiency was monitored by western blot analysis. (Lower panel) The expression levels of FASN and SREBP-1 in SIRT-1 knockdown cells were examined after gAcrp treatment. E MCF-7 cells were pretreated with EX527 for 2 h, followed by further incubation with gAcrp (1 μg/mL) for 12 h. mRNA levels of FASN, ACC-1, ACLY, and FADS2 were measured by RT-qPCR. F-H MCF-7 cells were transfected with SIRT-1 siRNA (25 nM) for 36 h, followed by treatment with 1 μg/mL of gAcrp for 24 h ( F ), 48 h ( G ), or 6 h ( H ). F Cellular neutral lipid content was determined by Bodipy 493/503 uptake assay. G Cells were labeled with Alexa fluor 488-conjugated CT-B and lipid raft microdomains were observed under a confocal microscope. H Lipid droplets were stained with Nile red and autophagosomes were labeled with an Alexa fluor 488-conjugated anti-LC3 antibody. The overlapping between lipid droplets (red) and autophagosomes (green) were observed under a confocal microscope. The Mander’s overlap coefficient was used to test the colocalization of lipid droplets and autophagosomes. Scale bar: 20 μm. I-M MCF-7 cells were transfected with SIRT-1 siRNA (25 nM) ( I ) or pretreated with EX527 for 2 h ( J - M ), followed by further treatment with 1 μg/mL of gAcrp for 48 h. I and J Cell viability was measured by MTS assay. K - L The apoptosis level was determined using caspase-3/7 activity ( K ) and TUNEL assay ( L ) as indicated in the methods. M Expression levels of Bax and Bcl2 were examined by western blot analysis. * denotes p < 0.05 compared to control; # denotes p < 0.05 compared to cells treated with gAcrp alone; n=3 except where specifically indicated in Figures

Article Snippet: The specific transcription factor DNA binding activity was examined using the SREBP-1 Transcription Factor Assay Kit from Abcam (#ab133125) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Incubation, Transfection, Quantitative RT-PCR, Labeling, Microscopy, Staining, MTS Assay, Activity Assay, TUNEL Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Involvement of SIRT-1 induction and mTOR signaling in the modulation of SREBP-1 by globular adiponectin. A-D MCF-7 cells were treated with gAcrp (1 μg/mL) for 8 h ( A - B ) or transfected with 25 nM SIRT-1 siRNA for 36 h before treatment with gAcrp for 8 h. ( A and C ) Acetylated SREBP-1 level was determined by immunoprecipitation with an anti-acetyl lysine antibody and western blot analysis using an anti-SREBP-1 primary antibody. B and D Ubiquitinated SREBP-1 level was measured by immunoprecipitation with an anti-ubiquitin antibody, followed by western blot analysis using an anti-SREBP-1 primary antibody. E-F Cells were treated with gAcrp (1 μg/mL) for different time periods. The total and phospho-mTOR levels were determined by immunoblotting. G-H MCF-7 cells were transfected with SIRT-1 siRNA for 36 h ( G ) or pretreated with EX527 for 2 h ( H ), followed by treatment with gAcrp for additional 3 h. p-mTOR/mTOR levels were examined by western blot analysis. I-J MCF-7 cells were treated with different concentrations of rapamycin ( I ) and MHY1485 ( J ) for 8 h. SREBP-1 and FASN expression levels were measured by western blot analysis. K-L MCF-7 cells were treated with rapamycin and MHY1485 for 24 h. Intracellular lipid accumulation ( K ) and free fatty acid ( L ) levels were determined as described above. * indicates p < 0.05 compared to control; # indicates p < 0.05 compared to gAcrp-treated cells; n=3

Article Snippet: The specific transcription factor DNA binding activity was examined using the SREBP-1 Transcription Factor Assay Kit from Abcam (#ab133125) according to the manufacturer’s protocol.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Role of SIRT-1 in adiponectin modulation of in vivo breast tumor lipid metabolism and growth. MDA-MB-231-luc orthotopic breast tumor s were generated in BALB/c nude mice, followed by treatment with gAcrp alone or gAcrp in combination with EX527 for 28 days. A and B Luminescent images of tumors ( A ) and tumor growth rate were monitored by luminescent in vivo imaging during treatment ( B ). C and D Tumor tissues were harvested after 4 weeks of treatment. Isolated tumors were captured at the end of experiment ( C ) and tumor weight was recorded ( D ). E Tissue section was prepared, and cleavage of caspase-3 was examined by immunohistochemistry (IHC). The percentage of cleaved caspase-3 positive tumor cells was determined by Image J software. F The expression levels of Bax and Bcl2 were measured by western blot analysis. The representative images from 3 mice each group were shown along with blot quantification for all collected tumor tissues. G The expression levels of FASN and SREBP-1 were analyzed by immunoblotting analysis. H SREBP-1 was detected in tumor tissues by IHC. The proportion of nuclear SREBP-1 positive cells were presented in bar diagram. Scale bar: 100 μm. I The mRNA levels of SREBP-1, FASN, ACC-1, FADS2, and ACLY in tumor tissues were measured by RT-qPCR. J The protein levels of p-mTOR, mTOR, β-catenin, and LC3I/II were determined by western blot analysis. K-L Single cells were isolated from tumor tissues by incubating with collagenase solution. K Tumor cells were incubated with Bodipy 493/503 for 15 min at 37 o C, followed by flow cytometry analysis. L The free fatty acid level was measured in tumor cells and normalized to tumor cell number

Article Snippet: The specific transcription factor DNA binding activity was examined using the SREBP-1 Transcription Factor Assay Kit from Abcam (#ab133125) according to the manufacturer’s protocol.

Techniques: In Vivo, Generated, In Vivo Imaging, Isolation, Immunohistochemistry, Software, Expressing, Western Blot, Quantitative RT-PCR, Incubation, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Proposed model for the modulation of tumor fatty acid metabolism by adiponectin. Adiponectin has been reported to potently suppress breast cancer growth. This study focuses on effects of adiponectin on tumor fatty acid metabolism and provides a novel mechanism for its breast cancer suppressing activity. SIRT-1 plays a central role in metabolic actions of adiponectin in breast cancer cells. On the one hand, SIRT-1 induction leads to downregulation of SREBP-1 by direct deacetylation and destabilization of nuclear SREBP-1 or by suppressing SREBP-1 expression through inhibition of mTOR signaling. SREBP-1 suppression leads to decreased expression of key enzymes in fatty acid synthesis (FAS) pathway and resultant blockage of FAS. On the other, SIRT-1 induction by adiponectin stimulates lipophagy to degrade lipid droplets and promote utilization of fatty acids for energy production via fatty acid oxidation (FAO). Inhibition of FAS accompanied by elevated FAO result in impairment in cellular fatty acid pool, which in turn causes disruption of lipid rafts and raft-dependent signal transduction, and cell apoptosis as a final consequence

Article Snippet: The specific transcription factor DNA binding activity was examined using the SREBP-1 Transcription Factor Assay Kit from Abcam (#ab133125) according to the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, Inhibition, Transduction

Journal: Cancer research

Article Title: Syndecan-1 (CD138) suppresses apoptosis in multiple myeloma by activating IGF1 receptor: prevention by synstatin IGF1R inhibits tumor growth

doi: 10.1158/0008-5472.CAN-16-0232

Figure Lengend Snippet: A. CAG cells grown for 24 hr in complete culture medium containing 1–30 μM SSTNIGF1R are analyzed using ApoTox-GLO to quantify % cell viability or apoptosis compared to untreated cells; B. Percent of apoptotic cells (untreated vs. 30 μM SSTNIGF1R from part (A)) quantitated by TUNEL assay; C. CAG and MM.1R myeloma cells, MDA-MB-231 breast carcinoma and HUVEC vascular endothelial cells were grown for up to 24 hr in the presence or absence of 30 μM SSTNIGF1R. Cell lysates at various time points are assessed using an antibody array to analyze the relative expression and/or phosphorylation levels of stress and apoptotic markers. D. MM.1R and CAG myeloma cells are grown for 9 hr in complete culture medium and treated with vehicle (DMSO) alone or JNK inhibitor (JNK-IN-8) in the presence or absence of 30 μM SSTNIGF1R. Induction of apoptosis is quantified as fold increase in Caspase 3/7 activity (Caspase3/7-GLO) relative to vehicle alone (set to 1.0).

Article Snippet: The PathScan Stress and Apoptosis Signaling Antibody Array Kit was obtained from Cell Signaling Technology.

Techniques: TUNEL Assay, Ab Array, Expressing, Phospho-proteomics, Activity Assay

Journal: Cancer research

Article Title: Syndecan-1 (CD138) suppresses apoptosis in multiple myeloma by activating IGF1 receptor: prevention by synstatin IGF1R inhibits tumor growth

doi: 10.1158/0008-5472.CAN-16-0232

Figure Lengend Snippet: CAG myeloma cells implanted subcutaneously in immunodeficient nude (nu/nu) mice and are allowed to form palpable tumors for 7–10 days. Mice are then implanted with Alzet pumps delivering 0.25 μL/hr of 4 mM SSTNIGF1R, SSTNIGF1R-T or saline alone for an additional 4 weeks. A. Tumors harvested at the end of the 4-week treatment period (8 per cohort shown; ‘X’ denotes no tumor found). B. Quantification of tumor volume. Treated and untreated cohorts were compared using unpaired one-tail t-test. P-values of less than 0.05 were considered significant (**). Data represent mean plus or minus SEM. C. Representative tumors sectioned and stained with MECA-32 antibody specific for the pan-endothelial cell marker PV1/PLVAP. D. Quantification of overall MECA-32 staining intensity. E. Model depicts capture of inactive αvβ5 integrin and IGF1R via a docking site in the extracellular domain of Sdc1 (21,23), which causes autophosphorylation of IGF1R and binding of inactive ASK1 (with inactivating phosphorylation on Ser83, Ser966 (S) and tyrosines (Y)). Displacement of the integrin and IGF1R from Sdc1 inactivates IGF1R, allow ASK1 activation via autophosphorylation of Thr838 (T), and downstream activation of JNK via MKK4 or MKK7, leading to caspase activation and apoptosis.

Article Snippet: The PathScan Stress and Apoptosis Signaling Antibody Array Kit was obtained from Cell Signaling Technology.

Techniques: Saline, Staining, Marker, Binding Assay, Phospho-proteomics, Activation Assay

Journal: PLoS Genetics

Article Title: Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

doi: 10.1371/journal.pgen.1005305

Figure Lengend Snippet: ( A-B ) Graphs showing mean liver size ± SEM of 6-day-old control sibling and transgenic zebrafish larvae treated for 3 days with amitriptyline ( A ) or paroxetine ( B ) at the indicated dosages. N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing drug-treated zebrafish to DMSO-treated siblings with the same genotype: *, p<0.05; **, p<0.01; ***, p<0.001. ( C ) Graph showing mean liver size ± SEM of 6-day-old control sibling and transgenic zebrafish larvae treated for 3 days with 0.5% DMSO (-), 20 μM amitriptyline (AMI), 2 μM SP600125 (SP), or both drugs combined (AMI+SP). N values are shown above the x-axis. Asterisks indicate p-values for 2-way ANOVA comparing each group of transgenic zebrafish to AMI+SP group: **, p<0.01; ***, p<0.001. N.S., no significant difference between groups of non-transgenic zebrafish (2-way ANOVA). ( D ) Representative images of control, non-hydrodynamically transfected (non-HDT) mouse livers (left) and mouse liver tumors induced by hydrodynamic transfection of activated β-catenin and Met (Met/β-cat HDT). Mice were treated with saccharine alone (vehicle only, top row) or amitriptyline plus saccharine (bottom row). Scale bars, 1 cm. ( E ) Graph showing mean liver-to-body mass ratios ± SEM for non-HDT and HDT-Met/β-cat mice treated with saccharine alone (vehicle) or amitriptyline plus saccharine (+Ami). P values calculated with Mann-Whitney test. N values are shown above the x-axis. ( F ) Representative hematoxylin-and-eosin-stained, Ki-67-labeled, and TUNEL-labeled images from vehicle- and amitriptyline-treated mice. Ki-67 and TUNEL stainings were performed using 3, 3'-diaminobenzidine (DAB) substrate, so positive-staining cells are brown, and hematoxylin counterstain to highlight nuclei and other basophilic structures in blue. ( G-H ) Graphs showing mean ± SEM of Ki-67-positive ( G ) or TUNEL-positive ( H ) cells per high-power field. P values calculated with Mann-Whitney test. N values are shown above the x-axis.

Article Snippet: Samples were detected with VECTASTAIN Elite ABC Reagent (Vector Labs) and Vector DAB substrate kit (Vector Labs) and counterstained with hematoxylin.

Techniques: Transgenic Assay, Transfection, MANN-WHITNEY, Staining, Labeling, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 1. Neutrophils accumulate in the hippocampus and contribute to the progression of SAE. (A and B) Representative immunoblots and quantification of Ly6G levels in the hippocampus at 24 hours after operation. (C and D) Representative FACS plots and quantification of neutrophils (Ly6G+CD11b+) measured by flow cytometry in blood at 24 hours after operation. (E and F) Freezing to context and freezing to tone examined at 24 hours after operation. (G) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (H) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (I) The quantitative results of the percentage of TUNEL-positive area in the total area of the image (whole microscopic field) in the hippocampus. (J) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (K) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6 for Fig. B, D, G, I and K; n=8 for Fig. E and F; **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Western Blot, Flow Cytometry, Permeability, TUNEL Assay, Immunofluorescence, Staining

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 2. Sepsis induces neutrophil releasing NETs and NETs digestion with DNase I attenuates the development of SAE. (A) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (B) Quantification of the percentage of Cit-H3-positive neutrophils. (C) Levels of plasma cfDNA are measured at 24 hours after operation. (D and E) Representative immunoblots of NETs appearance (D) and quantification of the Cit-H3 levels (E) in the hippocampus at 24 hours after operation. (F) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) Total NETs score of each group. (H and I) Freezing to context and freezing to tone examined at 24 hours after operation. (J) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (K) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (L) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (M) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures, and the scale bar indicates 10 μm. (N) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence, Permeability, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 3. GSDMD regulates NET release and GSDMD deficiency in neutrophils attenuates the progression of SAE. (A) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (B) Quantification of the percentage of Cit-H3-positive neutrophils. (C) Levels of plasma cfDNA are measured at 24 hours after operation. (D and E) Representative immunoblots of NETs appearance (D) and quantification of the Cit-H3 levels (E) in the hippocampus at 24 hours after operation. (F) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) Total NETs score of each group. (H and I) Freezing to context and freezing to tone examined at 24 hours after operation. (J) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (K) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (L) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (M) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (N) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence, Permeability, TUNEL Assay

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 4. PD-L1 can regulate GSDMD expression in septic neutrophils. (A to C) Representative immunoblots and quantification of PD-L1 and GSDMD levels in the neutrophils from healthy subjects and septic patients. (D to F) Representative immunoblots and quantification of PD-L1 and GSDMD levels in the neutrophils from septic patients at 21 hours after PD-L1 siRNA treatment. (G) The GSDMD mRNA levels of neutrophils from septic patients at 21 hours after PD-L1 siRNA treatment. (H) Representative immunofluorescence images of isolated peripheral blood neutrophils from healthy subjects and septic patients. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (I) Quantification of the percentage of Cit-H3-positive neutrophils. (J) cfDNA levels of supernatant of cultured neutrophils are measured at 21 hours after PD-L1 siRNA treatment. (K) The nucleus is extracted from neutrophils from healthy subjects and septic patients. Representative immunoblot of PD-L1 in neutrophil nucleus. (L) Representative images of neutrophils PD-L1 from healthy subjects and septic patients by confocal microscopy. Neutrophils are stained with PD-L1 (green) and DAPI (blue). Scale bar indicates 10 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 5 μm. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant, 2-tailed Student’s t test for 4B, 4C, 4E, and 4F; one-way analysis of variance for 4G, 4I, 4J).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, Western Blot, Immunofluorescence, Isolation, Staining, Cell Culture, Confocal Microscopy

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 5. nPD-L1 forms a complex with p-Y705-Stat3 to transcriptionally activate GSDMD expression in neutrophils from septic patients. (A and B) Representative immunoblots and quantification of p-Y705-Stat3 level in the neutrophils from healthy subjects and sepsis patients. (C) Immunoprecipitation (IP) and western blot analysis of the PD-L1/p-Y705-Stat3 interaction in neutrophils from healthy subjects and septic patients. (D) Representative images of neutrophils PD-L1/p-Y705-Stat3 interaction from healthy subjects and septic patients by confocal microscopy. Neutrophils are stained with p-Y705-Stat3 (green), PD-L1 (red) and DAPI (blue). Scale bar indicates 10 μm. Higher magnification images are shown at the right row of figures, and the scale bar indicates 5 μm. (E and F) The nucleus is extracted from septic neutrophils at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). Representative immunoblots and quantification of PD-L1 level in neutrophil nucleus. (G and H) Representative immunoblots and quantification of GSDMD level in the neutrophils from septic patients at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). (I) The GSDMD mRNA levels of neutrophils from septic patients at 24 hours after treating with DMSO or inhibitor HO-3867 (20 μM). (J) Sequential ChIP-PCR analysis of the interactions between p-Y705-Stat3 and the GSDMD promoter in septic neutrophils. The values are presented as mean ± SD (n=6; **P<0.01, ****P<0.0001, ns=not significant, 2-tailed Student’s t test for 5B; one-way analysis of variance for 5F, 5H, and 5I).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, Western Blot, Immunoprecipitation, Confocal Microscopy, Staining

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 6. Genetic deletion of neutrophil PD-L1 reduces the expression of GSDMD in neutrophils in vivo and attenuates the release of NETs in CLP mice. (A and B) Representative FACS plots and quantification of GSDMD+ neutrophils measured by flow cytometry in blood at 24 hours after operation. (C) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils were stained with GSDMD (green) and DAPI (blue). Scale bar indicates 20 μm. Arrows indicate GSDMD+ neutrophils. (D) Quantification of the percentage of GSDMD-positive neutrophils. (E) Representative immunofluorescence images of isolated peripheral blood neutrophils at 24 hours after operation. Neutrophils are stained with SYTOX Orange (red) and Cit-H3 (green). Arrows indicate NETs. Scale bar indicates 10 μm. (F) Quantification of the percentage of Cit-H3-positive neutrophils. (G) Levels of plasma cfDNA are measured at 24 hours after sham or CLP surgery. (H and I) Representative immunoblots of NETs appearance (H) and quantification of the Cit-H3 levels (I) in the hippocampus at 24 hours after operation. (J) Representative immunofluorescence images of Cit-H3 (green) and MPO (red) staining with blue DAPI nuclear staining in hippocampus. Neutrophils express MPO (red) and NET forming neutrophils also express Cit-H3 (green). Cyan fluorescence represents the colocalization of Cit-H3 with DNA. The white arrows point to neutrophils with NETs and the red arrows to neutrophils without NETs. The scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (K) Total NETs score of each group. The values are presented as mean ± SD (n=6; ****P<0.0001, ns=not significant, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Expressing, In Vivo, Flow Cytometry, Immunofluorescence, Isolation, Staining, Clinical Proteomics, Western Blot, Fluorescence

Journal: International journal of biological sciences

Article Title: PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

doi: 10.7150/ijbs.79913

Figure Lengend Snippet: Figure 7. Genetic knockout of Neutrophil PD-L1 attenuates the progression of SAE. (A and B) Freezing to context and freezing to tone examined at 24 hours after operation. (C) The BBB permeability of hippocampus evaluated by Evans blue extravasation at 24 hours after operation. (D) Representative TUNEL (green) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (E) The quantitative results of the percentage of TUNEL positive area in the total area of the image (whole microscopic field) in the hippocampus. (F) Representative IBA-1 (red) and DAPI (blue) immunofluorescence staining in the hippocampus. Scale bar indicates 20 μm. Higher magnification images are shown at the right row of figures-scale bar indicates 10 μm. (G) The quantitative results of the percentage of IBA-1 positive area in the total area of the image (whole microscopic field) in the hippocampus. (H) Schematic illustration of the main findings: Sepsis-induced NETosis contributes to hippocampus-dependent memory impairment, and increases BBB permeability, neuronal apoptosis, and microglia activation in the hippocampus region. The NET release is promoted by the cleavage of GSDMD, which is transcriptionally regulated by the nuclear translocation of a PD-L1/p-Y705-Stat3 complex. Together, PD-L1/Stat3/GSDMD is essential for NET production and development of sepsis-associated encephalopathy. The values are presented as mean ± SD (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way analysis of variance).

Article Snippet: Mice blood neutrophils were isolated by positive selection magnetic cell separation (MACS) using the Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec, Bergisch Gladbach, Germany) according to instructions.

Techniques: Knock-Out, Permeability, TUNEL Assay, Immunofluorescence, Staining, Activation Assay, Translocation Assay

Journal: Science Advances

Article Title: Gain-of-function mutations in the catalytic domain of DOT1L promote lung cancer malignant phenotypes via the MAPK/ERK signaling pathway

doi: 10.1126/sciadv.adc9273

Figure Lengend Snippet: ( A ) DOT1L -depleted NSCLCs were infected with lentivirus encoding doxycycline (Dox)–inducible WT or mutant DOT1L (Tet-on system). ( B ) The cells generated in (A) were subjected to clonogenic survival assay by crystal violet staining 7 to 14 days after seeding. ( C ) The cells generated in (A) were seeded for tumorsphere formation assay and cultured for 7 to 14 days. Scale bars, 1000 μm. ( D ) Effect of doxycycline induction on the relative tumor volume in BALB/c-nu mice xenografted with NCI-H460-DOT1L-KD-DOT1L-WT/R231Q cells ( n = 10 mice per group). ( E ) Survival analysis of mice in the DOT1L WT and R231Q groups ( n = 20 mice per group and tumor volume > 2000 mm 3 ). ( F ) Western blotting analysis of DOT1L-flag and H3K79me2 expression in xenograft tumors from the CDX model in (D) ( n = 3). ( G ) Ki67 staining and TUNEL staining of the CDX tumors. Scale bars, 50 μm. Data are shown as means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. In (A), P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. Data from (B) to (D), (F), and (G) were analyzed using Student’s t test (independent samples t test). The log-rank (Mantel-Cox) test was used for (E), ** P < 0.01.

Article Snippet: Apoptosis in tumor sections of xenograft models was detected using the TUNEL Assay Kit-HRP-DAB (Abcam) according to the manufacturer’s protocol.

Techniques: Infection, Mutagenesis, Generated, Clonogenic Cell Survival Assay, Staining, Tube Formation Assay, Cell Culture, Western Blot, Expressing, TUNEL Assay

Journal: Science Advances

Article Title: Gain-of-function mutations in the catalytic domain of DOT1L promote lung cancer malignant phenotypes via the MAPK/ERK signaling pathway

doi: 10.1126/sciadv.adc9273

Figure Lengend Snippet: ( A ) Crystal violet staining of NCI-H460-DOT1L-WT/R231Q cells treated with SGC0946 (2.5 or 7.5 μM), binimetinib (2.5 or 7.5 μM), or the combination at the same concentrations for 7 to 14 days (top). Comparison of relative colony formation between groups at 7.5 μM drug concentration (bottom). ( B ) Clonogenic survival assay to evaluate the effects of DOT1Lis SGC0946 (1 μM), binimetinib (1 μM), or the combination at the same concentrations on cells transfected with constructs expressing the other DOT1L gain-of-function mutants (Y216C, S225L, N241T, F243L, and A1003G). The plates were photographed 7 to 14 days after seeding. ( C ) Tumor growth curves and images for four xenograft models ( n = 6 mice per group) that were treated with vehicle (10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline, five times per week), SGC0946 (10 mg/kg, five times per week), binimetinib (15 mg/kg, five times per week), or the combination at the same doses through intraperitoneal administration and oral gavage. The graphs show the TIR%. ( D ) IHC staining for Ki67 and TUNEL staining of the NCI-H1299-DOT1L-R231Q CDX tumor tissues. Scale bars, 50 μm. ( E ) Levels of RAF1, DOT1L-flag, p-MEK, MEK, and H3K79me2 in tumors from the NCI-H1299-DOT1L-R231Q CDX mice ( n = 2). ( F ) Summary diagram describing that regulation gain-of-function DOT1L mutations increase the malignant phenotypes of lung cancer by regulating the MAPK/ERK signaling pathway. Data are shown as means ± SEM. * P < 0.05, as compared to the WT group. # P < 0.05, ## P < 0.01, and ### P < 0.001, as compared to the single treatment group. In (A), P values were determined using one-way ANOVA with Tukey’s multiple comparisons test and Student’s t test (independent samples t test). In (C) and (D), P values were determined using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Apoptosis in tumor sections of xenograft models was detected using the TUNEL Assay Kit-HRP-DAB (Abcam) according to the manufacturer’s protocol.

Techniques: Staining, Comparison, Concentration Assay, Clonogenic Cell Survival Assay, Transfection, Construct, Expressing, Saline, Immunohistochemistry, TUNEL Assay

Journal:

Article Title: Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

doi: 10.1172/JCI23203

Figure Lengend Snippet: Efficient adenoviral delivery of phosphorylation-deficient survivin (Ad-GFP-S-M) to PASMCs in vitro reduces proliferation and increases apoptosis. (A) Primary culture of rat PASMCs stains positive for smooth muscle actin but not vWF, indicating no contamination with endothelial cells. (B) Infection of PASMCs with adenoviruses encoding GFP and either WT survivin (Ad-GFP-S) or survivin mutant (Ad-GFP-S-M) was highly efficient, as evidenced by GFP reporter (green fluorescence in the left panels and differential interference contrast [DIC] in the right panels). Note the reduced cellularity of the plate infected with Ad-GFP-S-M, compared with Ad-GFP-S. (C) Cells infected with Ad-GFP-S-M (grown in 10% FBS) show increased TUNEL-positive nuclei and reduced PCNA-positive nuclei, suggesting that they undergo apoptosis and not proliferation. In contrast, cells infected with Ad-GFP-S (grown in 0.1% FBS) show no apoptosis and increased rates of PCNA expression. (D) Mean data for TUNEL- and PCNA-positive nuclei, 48 hours after infection with Ad-GFP-S-M versus Ad-GFP-S; 5 fields studied in each plate, 20 plates per group. *P < 0.01.

Article Snippet: ApopTag apoptosis detection kit (TUNEL stain, Serologicals Corp.) and the PCNA antibody (Dako-Cytomation) were used according to the manufacturer’s instructions.

Techniques: In Vitro, Infection, Mutagenesis, Fluorescence, TUNEL Assay, Expressing

Journal:

Article Title: Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

doi: 10.1172/JCI23203

Figure Lengend Snippet: Ad-GFP-S-M infection induces PASMC mitochondria-dependent apoptosis. (A) PASMCs effectively infected with Ad-GFP-S (green) show slight hyperpolarization of mitochondrial membrane potential (increased TMRM fluorescence) compared with noninfected cells. In contrast, mitochondria of Ad-GFP-S-M–infected cells (but not neighboring noninfected cells) are less red, indicating depolarized mitochondria. Mean data are shown on the right (arbitrary fluorescence units [FU], means from 15 plates per group; *P < 0.01). Immunoblots show that, in contrast to expression of WT survivin, expression of survivin mutant in PASMCs grown in 10% FBS induces activation of caspase-9 and caspase-3. (B) PASMCs infected with Ad-GFP-S demonstrate sequestered cytochrome c within mitochondria, as shown by the punctate pattern of staining. In contrast, mitochondria of Ad-GFP-S-M–infected cells show cytochrome c–positive staining diffusely throughout the cell, indicating leakage of cytochrome c from mitochondria into the cytoplasm. Magnification: left and middle panels, ×75; right panels, ×125. (C) In contrast to infection with Ad-GFP-S, infection with Ad-GFP-S-M induces translocation of the mitochondria-based apoptosis-inducing factor (AIF) in the nucleus, where it initiates caspase-independent apoptosis. Magnification, ×100.

Article Snippet: ApopTag apoptosis detection kit (TUNEL stain, Serologicals Corp.) and the PCNA antibody (Dako-Cytomation) were used according to the manufacturer’s instructions.

Techniques: Infection, Fluorescence, Western Blot, Expressing, Mutagenesis, Activation Assay, Staining, Translocation Assay

Journal:

Article Title: Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

doi: 10.1172/JCI23203

Figure Lengend Snippet: Selective expression of survivin mutant in resistance PAs causes an increase in PASMC outward K+ current. (A) In FBS-rich conditions (10% FBS in the medium, a condition known to increase endogenous survivin), infection with Ad-GFP-S-M causes augmentation of K+ currents and decreased capacitance (Cm, a measure of cell size), consistent with apoptosis; the opposite is seen with Ad-GFP-S infection. In contrast, in serum-deprived conditions (0.1% FBS), infection with Ad-GFP-S causes a decrease in K+ currents, consistent with apoptosis resistance. Since in these conditions endogenous survivin is absent, infection with Ad-GFP-S-M has no effect on K+ current. Cells carrying the transgenes were selected by the green fluorescence. Mean data for current density over voltage are shown on the right (n = 6 cells per group; *P < 0.01 vs. control). (B) Both GFP immunofluorescence microscopy and quantitative RT-PCR of laser-capture-microdissected resistance PAs demonstrate efficient delivery of the transgenes, particularly to the very small (less than 50 μm) resistance PAs (arrows). (C) In our inhaled gene therapy approach, the expression of the transgenes is restricted to the lungs, as shown by the expression of GFP, measured by quantitative RT-PCR. The expression of endogenous survivin in nontreated rats is minimal in all organs studied, except the spleen. Our WT-survivin primer also detects the survivin mutant, as shown by the increased lung signal in the treated rats. Data from 5 rats per group are shown.

Article Snippet: ApopTag apoptosis detection kit (TUNEL stain, Serologicals Corp.) and the PCNA antibody (Dako-Cytomation) were used according to the manufacturer’s instructions.

Techniques: Expressing, Mutagenesis, Infection, Fluorescence, Immunofluorescence, Microscopy, Quantitative RT-PCR

Journal:

Article Title: Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

doi: 10.1172/JCI23203

Figure Lengend Snippet: Ad-GFP-S-M augments apoptosis and Kv current and reduces proliferation within resistance PAs in vivo. (A) The number of TUNEL-positive nuclei (arrows) is increased by Ad-GFP-S-M treatment, while the number of PCNA-positive nuclei is reduced, compared with those in the Ad-GFP–treated and nontreated MCT-PAH rats. *P < 0.05 vs. MCT; †P < 0.05 vs. Ad-GFP. (B) Reduced BrdU staining (green) in resistance PAs of rats treated with Ad-GFP-S-M, compared with Ad-GFP-S (a representative image from 5 rats per group is shown). (C) Freshly isolated PASMCs from rats treated with Ad-GFP-S-M have increased K+ currents, in agreement with our in vitro data (Figure ​(Figure5A).5A). The sensitivity to 4-aminopyridine (4-AP; 5 mM) and current morphology suggest that the induced current is voltage-gated (Kv). *P < 0.05 vs. Ad-GFP.

Article Snippet: ApopTag apoptosis detection kit (TUNEL stain, Serologicals Corp.) and the PCNA antibody (Dako-Cytomation) were used according to the manufacturer’s instructions.

Techniques: In Vivo, TUNEL Assay, BrdU Staining, Isolation, In Vitro