Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to HeLa cells. Following 2 h incubation the cells were lysed and the CNF1-catalysed deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting (A). HeLa cells were incubated with an anti Lu/BCAM antibody (AB B12) that binds to the extracellular domain or as control with an anti-Lu/BCAM antibody (AB C16) directed against the intracellular part of the glycoprotein. GST-CNF1 was then added to the cells for 2 h. We followed the toxins uptake by the amount of modified RhoA (shift in SDS-PAGE, B). Shown is a typical result of 3 independent experiments. Colocalization of DyLight488-labeled GST-CNF1 with Lu/BCAM and EEA1 (C) Top: HeLa-cells were treated on ice with DyLight488-labeled GST-CNF1 (5 mg/ml) (green) for 30 min to allow receptor binding. After 30 min cells were transferred to 37° for 30 min to induce uptake. Subsequently cells were fixed and stained for EEA1 (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a maximal projection of all confocal planes. Scale bar indicates 10 µm. Middle: HeLa-cells were treated with labeled GST-CNF1 as in A. After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Bottom: HeLa-cells were treated as in A, but instead of GST-CNF1, cells were treated with DyLight488-labeled GST-CNFY (5 mg/ml) (green). After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Shown is a typical staining of 9 HeLa cells analyzed.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Incubation, Recombinant, Modification, SDS Page, Western Blot, Labeling, Binding Assay, Staining, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: A) K562 leukemia cells, which do not express Lu/BCAM (top) and the isogenic cell line K562-Lu/BCAM expressing the receptor (bottom), were treated with GST-CNF1 for different time periods from 1 h to overnight (ON) as indicated. Uptake of the toxin was analyzed by the shift of modified RhoA in SDS-PAGE. B) GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to K562-Lu/BCAM cells for 2 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting. Data are representative for at least 3 independent experiments.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Expressing, Modification, SDS Page, Incubation, Recombinant, Western Blot

Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: A) For dot-blots 5 µl of 3 µM solutions of GST-CNFs, GST-CNF fragments and GST alone, respectively, were spotted onto a nitrocellulose membrane. The membrane was blocked with skimmed milk and recombinant BCAM (6 µM) was added for 1 h at room temperature. Following washing bound rBCAM was detected with an anti-Lu/BCAM antibody. Equal protein load was analyzed by visualizing the GST part of the spotted proteins with an anti GST-antibody. B) Biacore protein-protein interaction studies: An antibody against human IgG (Millipore) was coupled to two lanes of a CM5-biacore chip. As ligand recombinant BCAM containing a C-terminal human IgG domain (Sino biologics) was exclusively guided over lane 2. In a second step, GST-CNF proteins as analyte were guided over both lanes. Bound protein is given as relative units (RU) corrected for the unspecific binding to lane 1 as average plus standard deviation of three independent experiments.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Recombinant, Binding Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Age-related decline of hemoglobin-α (Hb-α) with parallel increase in brain hypoxia in the intact male mouse brain. ( A ) Representative photomicrographs from male mouse hippocampal CA1, CA3 and cerebral cortex regions showing age-related decline in Hb-α staining and parallel increase in brain hypoxia as measured by pimonidazole (hypoxyprobe-1) staining. ( B ) Semi-quantitative intensity analysis of Hb-α and pimonidazole staining in hippocampal CA1 and CA3 regions and cerebral cortex of all male mice at different ages. ( C ) Correlation coefficient analysis between Hb-α and pimonidazole in hippocampal CA1, CA3 and cortex regions. *: p < 0.05 vs. 6 months; #: p < 0.05 vs. 12 months; n = 5. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Age-related decline of hemoglobin-α (Hb-α) with parallel increase in brain hypoxia in the intact female mouse brain. ( A ) Representative photomicrographs from female mouse hippocampal CA1, CA3, and cerebral cortex regions showing age-related decline in Hb-α staining and parallel increase in brain hypoxia as measured by pimonidazole (hypoxyprobe-1) staining. ( B ) Semi-quantitative intensity analysis of Hb-α and pimonidazole staining in hippocampal CA1 and CA3 regions and cerebral cortex of all female mice at different ages. ( C ) Correlation coefficient analysis between Hb-α and pimonidazole in hippocampal CA1, CA3 and cortex regions. *: p < 0.05 vs. 6 months; #: p < 0.05 vs. 12 months; n = 5. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Gender differences in neuronal hemoglobin-α (Hb-α) levels and brain hypoxia in the mouse brain in aging. Semi-quantitative intensity analysis of Hb-α ( A – C ) and pimonidazole staining ( D – F ) in hippocampal CA1 and CA3 regions and cerebral cortex of all female and male mice at different ages. *: p < 0.05 vs. female; n = 5.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on HIF-1α expression in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A )/( a ) and ( B )/( a ) Representative photomicrographs of HIF-1α immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. ( A )/( b ) and ( B )/( b ) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in HIF-1α expression in sham after Hb-α knockdown (*: p < 0.05, n = 6). At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased HIF-1α expression in both hippocampal CA1 ( A ) and CA3 regions ( B ). (*: p < 0.05, **: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on expression of the HIF-1α-regulated pro-apoptotic gene, PUMA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A , C ) Representative photomicrographs of PUMA immunostaining in 3-month-old male mouse hippocampal CA1 ( A ) and CA3 ( C ) regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. Staining in red represents NeuN, a neuronal marker, and staining in green represents PUMA. ( B , D ) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in PUMA intensity in sham after Hb-α knockdown. At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased PUMA intensity in the hippocampal CA1 and CA3 regions (**: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Staining, Marker, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on expression of the HIF-1α-regulated pro-apoptotic gene, NOXA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A , C ) Representative photomicrographs of NOXA immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. ( B , D ). Quantification of mean immuno-fluorescence (IF) demonstrates a mild increase in NOXA expression in sham after Hb-α knockdown (**: p < 0.01, n = 6). At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased NOXA expression in both CA1 ( A ) and CA3 regions ( C ). (**: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on neurodegeneration in the male mouse hippocampal CA1 region following global cerebral ischemia (GCI). ( A ) Representative photomicrographs from male mouse hippocampal CA1 region showing staining with the neuronal marker NeuN or with the neurodegeneration marker Fluoro Jade C (FJC). ( B ) NeuN-positive cell number in hippocampal CA1 region of various groups. ( C ) Intensity analysis of FJC staining in hippocampal CA1 region of various groups. **: p < 0.01. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on neurodegeneration in the male mouse hippocampal CA3 region following global cerebral ischemia (GCI). ( A ) Representative photomicrographs from male mouse hippocampal CA3 region showing staining with the neuronal marker NeuN or with the neurodegeneration marker Fluoro Jade C (FJC). ( B ) NeuN-positive cell number in hippocampal CA3 region of various groups. ( C ) Intensity analysis of FJC staining in hippocampal CA3 region of various groups. *: p < 0.05, **: p < 0.01. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Staining, Marker

Journal: Poultry Science

Article Title: Primary growth plate chondrocyte isolation, culture, and characterization from the modern broiler

doi: 10.1016/j.psj.2022.102254

Figure Lengend Snippet: Evaluation of protein production and secretion by primary chondrocytes in culture. Protein expression for COLI, COLII, COLXA1, ACAN, Sox9, and GAPDH in cell lysate and media in addition to Ponceau S stain of the media blot and subsequent statistical analysis in culture (A, B, C). Significance was determined using a one-way ANOVA with P -value < 0.05. (B) $ indicates significant difference between Sox9 expression on d14, d18, and d21 compared to d3. # indicates significant difference between COLI expression on d18 compared to d3, d7, and d11. (C) + indicates significant difference between ACAN secretion on d11, d14, d18, and d21 compared to d3. * indicates significant difference in COLII secretion on d7 compared to d3, d14, d18, and d21. (D) Immunofluorescence staining of COLI (594-conjugated) and COLII (488-conjugated), along with DAPI nuclear dye, in chondrocytes d7 and d18 in culture. ACAN, aggrecan; COL, collagen; Sox, SRY-Box.

Article Snippet: Primary antibodies used were rabbit anti-COLI (Origene, Rockville, MD), mouse anti-COLII (Novis Biologicals, Littleton, CO), rabbit anti-ACAN (ABClonal, Woburn, MA), rabbit anti-Sox9 (ABClonal, Woburn, MA), and rabbit anti-COL10A1 (ABClonal, Woburn, MA).

Techniques: Expressing, Staining, Immunofluorescence

Journal: The FASEB Journal

Article Title: Cystathionine β-synthase regulates mitochondrial morphogenesis in ovarian cancer

doi: 10.1096/fj.201701095R

Figure Lengend Snippet: CBS regulates MFN2 expression. A) Expression of mitochondrial fusion and fission markers MFN2, MFN1, Opa1, Drp1, and Fis1 were determined by immunoblotting in CP20 and OV90 cells, either transfected with scrambled siRNA (siCTL) or siRNA against the CBS gene for 72 h. Efficient gene silencing was determined by immunoblotting with the CBS antibody, and GAPDH served as a loading control. B) Immunoblotting of CBS, MFN2, and MFN1 from tumor tissue of orthotopically implanted control (shCTL) and CBS knockdown (shCBS) A2780 cells. GAPDH is used as a loading control. T1–T3, tumors 1-3. C) Mitochondrial morphology was depicted by immunofluorescence of siCTL and siCBS OV90 cells, stained with the Mitotracker Red CMXRos. Original scale bars, 10 µm. D) Representative images from each group stained with the mitochondrial marker COXIV were selected to illustrate the analysis process. Color channels were separated from each image so that only mitochondria (green) were present and then skeletonized for network analysis. Mitochondrial networks are defined as any segment with at least 1 additional branch (blue arrows), whereas individual mitochondria are defined as unbranched (yellow arrows). E) Images were analyzed using a publicly available ImageJ macro designed by Valente et al. (32). A Student’s t test (α = 0.05) was used to compare CBS knockdown siCBS (n = 16) with control siCTL (n = 20) CP20 cells. All experiments were performed 3 times in triplicates, and values are represented as mean fold change ± sd. P ≤ 0.05 is considered statistically significant.

Article Snippet: Reagents, cell lines, and culture The following antibodies were used for immunoblotting: rabbit polyclonal CBS antibody (H-300, sc-67154; Santa Cruz Biotechnology, Dallas, TX, USA), mouse β-actin mAb (A-2228; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal to MFN2 (D2D10 rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal MFN1 antibody (ab126575; Abcam, Cambridge, United Kingdom), rabbit polyclonal OPA1 antibody (ab42364; Abcam), rabbit polyclonal to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G9545; Sigma-Aldrich), and anti-DRP1 (ab56788; Abcam).

Techniques: Expressing, Western Blot, Transfection, Control, Knockdown, Immunofluorescence, Staining, Marker

Journal: The FASEB Journal

Article Title: Cystathionine β-synthase regulates mitochondrial morphogenesis in ovarian cancer

doi: 10.1096/fj.201701095R

Figure Lengend Snippet: Clinicopathological significance of CBS and MFN2 in cancer. A) Expression of CBS, MFN1/2, HUWE1, and Parkin in normal (OSE, HOSE, FTE) vs. OvCa cells, as determined by immunoblotting. B, C) Kaplan-Meier overall survival curves in CBS (B) and MFN2 (C) low- and high-expression (mRNA data) OvCa cases from The Cancer Genome Atlas database. The percent probability of survival is plotted vs. time since diagnosis in months. OS, overall survival. D) Panel represents scatterplot of CBS vs. MFN2 with overlaid linear regression line among tumor tissue samples. Spearman correlation coefficient is 0.155 (P = 0.003).

Article Snippet: Reagents, cell lines, and culture The following antibodies were used for immunoblotting: rabbit polyclonal CBS antibody (H-300, sc-67154; Santa Cruz Biotechnology, Dallas, TX, USA), mouse β-actin mAb (A-2228; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal to MFN2 (D2D10 rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal MFN1 antibody (ab126575; Abcam, Cambridge, United Kingdom), rabbit polyclonal OPA1 antibody (ab42364; Abcam), rabbit polyclonal to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G9545; Sigma-Aldrich), and anti-DRP1 (ab56788; Abcam).

Techniques: Expressing, Western Blot, Biomarker Discovery

Journal: The FASEB Journal

Article Title: Cystathionine β-synthase regulates mitochondrial morphogenesis in ovarian cancer

doi: 10.1096/fj.201701095R

Figure Lengend Snippet: CBS silencing increases oxidative stress and leads to MFN2 degradation by the ubiquitin-proteasome system. A) qRT-PCR was performed to determine relative mRNA expression of CBS, MFN1, and MFN2 in CP20 and OV90 cells transfected for 48 h with scrambled siRNA (siCTL) or siRNA against the CBS gene (siCBS). 36β4 was used as the internal control. B) CP20 cells were transfected as in A, and an equal amount of extracted protein was subjected to immunoprecipitation (IP) assay with MFN2 antibodies crosslinked with the agarose resin and further immunoprobed (IB) with ubiquitin and MFN2 antibodies. C) CP20 and OV90 cells were transfected with siCTL or siCBS and then treated with MG132 (5 µM/12 h) or left untreated. Expression of MFN2 and CBS was determined by immunoblot, and GAPDH was used to indicate equal protein loading. D) Total cellular ROS was determined by 2′,7′-dichlorofluorescein assay and quantified by fluorescence measurement in CP20 or OV90 cells transfected with scrambled siRNA (siCTL) or CBS siRNA (siCBS) and represented as relative fold change over control. E) CP20 and OV90 cells were transfected with siCTL or siCBS for 48 h and then treated with the antioxidant (Antx.; mitoTEMPO; 10 µM/12 h), and the expression of MFN2 and CBS was determined by immunoblotting. F) Expression of JNK, phosphorylated (p)p38, pERK1/2, and CBS was determined by immunoblotting of OV90 and CP20 cells, transfected for 72 h with siCTL or siCBS. G) CP20 and OV90 cells were transfected with siCTL or siCBS and then treated with SP600125 (SP; 20 µM/12 h) or left untreated. Expression of MFN2 and CBS was determined by immunoblot, and GAPDH was used to indicate equal protein loading. H) Equal amount of extracted protein from siCTL or siCBS CP20 cells were subjected to immunoprecipitation assay with MFN2 antibodies crosslinked with the agarose resin and further immunoprobed with HUWE1 (HUWE), CBS, p-Ser, and MFN2 antibodies. I) Equal amount of extracted protein from siCBS CP20 cells treated with vehicle or SP600125 (20 µM/12 h) were subjected to immunoprecipitation assay with MFN2 antibodies crosslinked with the agarose resin and further immunoprobed with p-Ser antibody. All experiments were performed 3 times in triplicates, and values are represented as mean fold change ± sd. *P ≤ 0.05 is considered statistically significant. J) Illustration depicts proposed mechanism of the CBS modulation of MFN2 stability. CBS regulates redox homeostasis, which if disrupted, activates the oxidative stress-sensing MAPK, JNK. Activated JNK phosphorylates MFN2, which recruits the E3 ligase, HUWE1, which in turn, ubiquitinates MFN2, thereby leading to its proteasomal degradation and mitochondrial fragmentation.

Article Snippet: Reagents, cell lines, and culture The following antibodies were used for immunoblotting: rabbit polyclonal CBS antibody (H-300, sc-67154; Santa Cruz Biotechnology, Dallas, TX, USA), mouse β-actin mAb (A-2228; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal to MFN2 (D2D10 rabbit mAb; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal MFN1 antibody (ab126575; Abcam, Cambridge, United Kingdom), rabbit polyclonal OPA1 antibody (ab42364; Abcam), rabbit polyclonal to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G9545; Sigma-Aldrich), and anti-DRP1 (ab56788; Abcam).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Expressing, Transfection, Control, Immunoprecipitation, Western Blot, Dichlorofluorescein Assay, Fluorescence

Journal: Cancer Science

Article Title: Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum‐based chemotherapy

doi: 10.1111/cas.15207

Figure Lengend Snippet: Immunohistochemical expression of Schlafen 11 (SLFN11) in bladder cancer (BC) and validation of the association between SLFN11 expression and clinical course. A, Representative immunohistochemical images of SLFN11 in BC. Scale bars, 200 µm (left) and 50 µm (right). B, Distribution of the immunohistochemical expression of SLFN11 in 120 BC cases, with 5% used as the cut‐off value. C, Correlation of the expression of SLFN11 protein with overall survival (OS) of 120 patients with BC. D, Correlation of the expression of SLFN11 protein with OS of 50 patients with unresectable locally advanced or metastatic BC treated with platinum‐based chemotherapy. E, Correlation of the expression of SLFN11 protein with OS of 70 patients with local BC treated by surgical resection without chemotherapy

Article Snippet: The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam).

Techniques: Immunohistochemical staining, Expressing

Journal: Cancer Science

Article Title: Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum‐based chemotherapy

doi: 10.1111/cas.15207

Figure Lengend Snippet: Association between Schlafen 11 (SLFN11) expression and clinicopathologic characteristics in bladder carcinoma (BC) patients (n = 120)

Article Snippet: The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam).

Techniques: Expressing

Journal: Cancer Science

Article Title: Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum‐based chemotherapy

doi: 10.1111/cas.15207

Figure Lengend Snippet: Univariate and multivariate analysis of factors for prognosis of bladder cancer patients treated with platinum‐based chemotherapy (n = 50)

Article Snippet: The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam).

Techniques: Expressing

Journal: Cancer Science

Article Title: Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum‐based chemotherapy

doi: 10.1111/cas.15207

Figure Lengend Snippet: Representative images of H&E and immunohistochemical staining. Images of H&E, Schlafen 11 (SLFN11), p53, GATA3, cytokeratin 5/6, and programmed death ligand 1 (PD‐L1) staining in bladder cancer cells. Scale bars, 50 µm

Article Snippet: The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam).

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum‐based chemotherapy

doi: 10.1111/cas.15207

Figure Lengend Snippet: Inactivation of Schlafen 11 (SLFN11) induces resistance to cisplatin and inhibits cell death under replication stress in bladder cancer (BC) cell lines. A, Correlation between SLFN11 mRNA expression and cisplatin sensitivity among 849 cell lines, which includes 15 BC cell lines in the Genomics of Drug Sensitivity in Cancer (GDSC) cancer cell line database. Pearson correlation ( r ) = .36, P = 1.1e‐27. B, Correlation between SLFN11 mRNA expression and cisplatin sensitivity among 15 BC cell lines with the GDSC cancer cell line database. r = .47, P = .075. C, Western blot analysis of SLFN11 in BC cell lines and β‐actin as a loading control. D, Western blot analysis of SLFN11 KO cells generated by CRISPR‐Cas9 gene editing technology. β‐Actin was used as a loading control. E, Immunohistochemical images of parent and SLFN11 KO cells in T24 or UM‐UC13 cell lines. Scale bars, 100 µm. F, Dose‐dependent effects of cisplatin on the viability of T24 or UM‐UC13 cell lines with parent and SLFN11 KO cells. G, Cell growth curves of the indicated cell lines under normal conditions (NT) or treated with the indicated concentrations of cisplatin for 4 h and released into a drug‐free medium. * P < .05; ** P < .01. act, drug activity (−log 10 [IC 50 M]); exp, mRNA expression (log 2 ); MGH, Massachusetts General Hospital; GDSC, Genomics of Drug Sensitivity in Cancer

Article Snippet: The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam).

Techniques: Expressing, Western Blot, Generated, CRISPR, Immunohistochemical staining, Activity Assay

Journal: Cancer Science

Article Title: Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum‐based chemotherapy

doi: 10.1111/cas.15207

Figure Lengend Snippet: Epigenetic modulators reactivate Schlafen 11 (SLFN11) expression and sensitize bladder cancer cells to platinum agents. A, Western blot analysis of RT112 (left) and 253JBV (right) cell lines treated with the indicated concentrations of 5‐aza‐2′‐deoxycytidine (5‐aza) or entinostat for 2 d. β‐Actin was used as a loading control. B, Representative immunohistochemical images of SLFN11 in RT112 (upper) and 253JBV (lower) cell lines treated with the indicated concentrations of 5‐aza or entinostat for 2 d. Scale bars, 100 µm. C, Dose‐dependent effects of cisplatin or carboplatin on the viability of RT112 (upper) and 253JBV (lower) cell lines treated with the indicated concentrations of 5‐aza or entinostat

Article Snippet: The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam).

Techniques: Expressing, Western Blot, Immunohistochemical staining

Journal: Science Advances

Article Title: Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson’s disease

doi: 10.1126/sciadv.abh1824

Figure Lengend Snippet: ( A ) Schematic of the screen. TH-Gal4–driven DUB-specific RNAi fly lines were used in an F1 primary screen to identify DUBs that rescued hPARIS-induced climbing defects on day 20. Such candidate DUBs were subjected to additional secondary F1 screens probing for DUBs that rescued climbing defects under conditions of DA parkin or PINK1 KD on day 20, respectively. Hits from the secondary screen were then examined for their ability to promote dopamine neuron survival in 20-day-old parkin or PINK1 KD flies. ( B ) Primary F1 screen based on rescue of PARIS-induced climbing defect identified 13 DUBs. ( C ) Summary of candidate DUBs from primary screen that rescued climbing defects in parkin KD flies. ( D ) Summary of candidate DUBs from primary screen that suppressed climbing defects in PINK1 KD flies and progressed for further validation. TH-Gal4/+ flies served as control. N = 60 flies per genotype for both primary and secondary screens. Quantitative data = means ± SEM. One-way analysis of variance (ANOVA); ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also figs. S1 and S2 and table S1.

Article Snippet: Primary antibodies used for immunoblot analyses include rabbit antibody to hPARIS (1:2000; Proteintech, catalog no. 24543-1-AP), rabbit antibody to Drosophila PARIS [1:1000; ( )], rabbit antibody to CYLD (1:1000, Cell Signaling Technology, catalog no. 8462), rabbit antibody to CYLD (1:1000; Sigma-Aldrich, SAB4200060), mouse antibody to parkin (Prk8) (1:1000; Cell Signaling Technology, catalog no. 4211), and rabbit antibody to PGC-1a (1:1000; Novus Biologicals, catalog no. NBP1-04676).

Techniques: Biomarker Discovery, Control

Journal: Science Advances

Article Title: Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson’s disease

doi: 10.1126/sciadv.abh1824

Figure Lengend Snippet: ( A ) Representative confocal image of the Drosophila whole brain showing the PPL1, PPL2, PPM1/2, and PPM3 DA neuron clusters. Scale bar, 100 μM. ( B ) Representative confocal images of individual dopamine neuron clusters visualized using TH immunofluorescence in the indicated genotypes under conditions of parkin KD. Scale bar, 50 μM. N = 10 flies per genotype. ( C ) Quantification of neuronal numbers within individual dopamine neuron clusters in the indicated genotypes. ( D ) Representative confocal images of individual dopamine neuron clusters visualized using TH immunofluorescence in the indicated genotypes under conditions of PINK1 KD or hPARIS overexpression. Scale bar, 50 μM. ( E ) Summary of dopamine neuron quantifications in the indicated genotypes. TH-Gal4/+ flies served as control. N = 10 flies per genotype. Quantitative data = means ± SEM. One-way ANOVA; **** P < 0.0001. See also fig. S3.

Article Snippet: Primary antibodies used for immunoblot analyses include rabbit antibody to hPARIS (1:2000; Proteintech, catalog no. 24543-1-AP), rabbit antibody to Drosophila PARIS [1:1000; ( )], rabbit antibody to CYLD (1:1000, Cell Signaling Technology, catalog no. 8462), rabbit antibody to CYLD (1:1000; Sigma-Aldrich, SAB4200060), mouse antibody to parkin (Prk8) (1:1000; Cell Signaling Technology, catalog no. 4211), and rabbit antibody to PGC-1a (1:1000; Novus Biologicals, catalog no. NBP1-04676).

Techniques: Immunofluorescence, Over Expression, Control

Journal: Science Advances

Article Title: Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson’s disease

doi: 10.1126/sciadv.abh1824

Figure Lengend Snippet: ( A ) Representative immunoblot and dPARIS quantification in flies expressing the indicated transgenes under the control of TH-Gal4 driver. TH-Gal4/+ flies served as control, N = 3. ( B ) Coimmunoprecipitation using anti-V5 antibodies shows interaction between C-terminal V5-tagged dPARIS and N-terminal Myc-tagged dCYLD in Drosophila S2 cells. Similar results were observed in three independent experiments. ( C ) Reciprocal coimmunoprecipitation experiments using anti-Myc antibodies verify dPARIS interaction with dCYLD in S2 cells transfected with indicated constructs in three independent experiments, N = 3. ( D ) Deubiquitination of dPARIS by dCYLD as assessed in S2 cells transfected with indicated constructs. Immunoblot analysis and relative quantification of immunoprecipitated (IP) dPARIS shows increased ubiquitination (Ub) of dPARIS in the presence of the dCYLD C284S catalytic mutant, thereby enhancing its proteasomal degradation, N = 3. ( E ) DUB activity of dCYLD affects the turnover rate of dPARIS. ( F ) Immunoblot analysis and dPARIS quantification in S2 cells at the indicated time points shows that while the dCYLD catalytic mutant accelerates dPARIS turnover, overexpression of WT dCYLD increases its half-life, N = 3. Quantitative data = means ± SEM. One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also fig. S4.

Article Snippet: Primary antibodies used for immunoblot analyses include rabbit antibody to hPARIS (1:2000; Proteintech, catalog no. 24543-1-AP), rabbit antibody to Drosophila PARIS [1:1000; ( )], rabbit antibody to CYLD (1:1000, Cell Signaling Technology, catalog no. 8462), rabbit antibody to CYLD (1:1000; Sigma-Aldrich, SAB4200060), mouse antibody to parkin (Prk8) (1:1000; Cell Signaling Technology, catalog no. 4211), and rabbit antibody to PGC-1a (1:1000; Novus Biologicals, catalog no. NBP1-04676).

Techniques: Western Blot, Expressing, Control, Transfection, Construct, Quantitative Proteomics, Immunoprecipitation, Ubiquitin Proteomics, Mutagenesis, Activity Assay, Over Expression

Journal: Science Advances

Article Title: Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson’s disease

doi: 10.1126/sciadv.abh1824

Figure Lengend Snippet: ( A ) Immunoblot analysis and quantification of endogenous PARIS in SH-SY5Y neuroblastoma cell line following CRISPR-Cas9–mediated KD or overexpression (OE) of CYLD, N = 3. ( B ) Cycloheximide chase experiment at the indicated time points showing PARIS turnover rate under conditions of CYLD KD or OE in SH-SY5Y cells. ( C ) Relative quantification of endogenous PARIS shows accelerated protein turnover under conditions of CYLD KD, whereas CYLD OE increases PARIS half-life. N = 3 independent experiments. ( D ) CYLD promotes deubiquitination of PARIS through hydrolysis of K48 ubiquitin chains. Ubiquitination of immunoprecipitated FLAG-tagged PARIS in SH-SY5Y cells monitored by immunoblot analysis under conditions of CYLD KD or OE in the presence of HA-tagged WT ubiquitin or ubiquitin mutants that can only append either K48 or K63 ubiquitin chains. ( E ) Quantification of PARIS ubiquitination showing enrichment of WT or K48 ubiquitin chains under conditions of CYLD KD but not CYLD OE. No changes in levels of K63 ubiquitin chains on PARIS observable under the different conditions assayed. N = 3. ( F ) GST pull-down assays using purified recombinant GST-tagged PARIS and WT CYLD indicate a robust interaction between PARIS and CYLD. Similar results were observed in three independent pull-down experiments. ( G ) Reciprocal GST pull-down assays using GST-tagged CYLD and PARIS confirm the direct interaction between PARIS and CYLD. N = 3. ( H ) In vitro deubiquitination of PARIS by CYLD. Immunoblot analysis of purified recombinant GST-PARIS ubiquitinated in the presence of PINK1 and activated parkin showing decreased ubiquitination in the presence of WT CYLD. The CYLD C601A catalytic mutant, however, has no impact on PARIS ubiquitination. Similar results were observed in three independent experiments. Quantitative data = means ± SEM. One-way ANOVA; * P < 0.05, ** P < 0.01, and **** P < 0.0001. See also fig. S5.

Article Snippet: Primary antibodies used for immunoblot analyses include rabbit antibody to hPARIS (1:2000; Proteintech, catalog no. 24543-1-AP), rabbit antibody to Drosophila PARIS [1:1000; ( )], rabbit antibody to CYLD (1:1000, Cell Signaling Technology, catalog no. 8462), rabbit antibody to CYLD (1:1000; Sigma-Aldrich, SAB4200060), mouse antibody to parkin (Prk8) (1:1000; Cell Signaling Technology, catalog no. 4211), and rabbit antibody to PGC-1a (1:1000; Novus Biologicals, catalog no. NBP1-04676).

Techniques: Western Blot, CRISPR, Over Expression, Quantitative Proteomics, Ubiquitin Proteomics, Immunoprecipitation, Purification, Recombinant, In Vitro, Mutagenesis

Journal: Science Advances

Article Title: Deubiquitinase CYLD acts as a negative regulator of dopamine neuron survival in Parkinson’s disease

doi: 10.1126/sciadv.abh1824

Figure Lengend Snippet: ( A ) Immunoblot analysis of the indicated proteins in control (H1) and two independent parkin knockout (P-KO1 and P-KO2) human midbrain dopamine neurons. ( B ) Quantification of indicated proteins in the different conditions. Mean values from three independent experiments shown. ( C ) Quantification of indicated mitochondrial markers in the different conditions. N = 3 independent experiments. ( D ) Quantification of TH-positive dopamine neurons in differentiated human midbrain cultures immunostained with TH and neuronal TUJ1 marker. N = 6 independent experiments ( E ) Immunoblot analysis of mitochondrial biomarkers in indicated conditions. ( F ) Quantification of indicated proteins under respective conditions. N = 3 independent experiments. ( G ) Immunoblot analysis and quantification of LC3II/I ratio in differentiated human midbrain neurons. N = 3 independent experiments. ( H ) Representative confocal images of human midbrain dopamine neurons immunostained for TH (green) and puromycin. Puromycin-labeled mitochondrial proteins detected by colocalization of anti-puromycin immunofluorescence (magenta) with the mitochondrial marker Tomm20 (red). ( I ) Quantification of anti-puromycin colocalization with Tomm20 in TH-positive midbrain dopamine neurons. Mean values from at least 15 TH-positive midbrain dopamine neurons from three independent differentiation shown. ( J ) Representative confocal images of human midbrain dopamine neurons expressing SNAP-Tag-Cox8a fusion protein immunostained for TH (magenta). Existing mitochondrial pool (old mito) shown in red and mitochondria newly assembled (new mito) shown in green. Nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). ( K ) Ratio of newly assembled to old mitochondria in TH-positive midbrain dopamine neurons. Mean values from at least 15 TH-positive midbrain dopamine neurons from three independent differentiations shown. ( L ) Immunoblot analysis and quantification of CYLD expression in SN from postmortem PD and age-matched controls. N = 4 per group. Quantitative data = means ± SEM. One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Primary antibodies used for immunoblot analyses include rabbit antibody to hPARIS (1:2000; Proteintech, catalog no. 24543-1-AP), rabbit antibody to Drosophila PARIS [1:1000; ( )], rabbit antibody to CYLD (1:1000, Cell Signaling Technology, catalog no. 8462), rabbit antibody to CYLD (1:1000; Sigma-Aldrich, SAB4200060), mouse antibody to parkin (Prk8) (1:1000; Cell Signaling Technology, catalog no. 4211), and rabbit antibody to PGC-1a (1:1000; Novus Biologicals, catalog no. NBP1-04676).

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

Journal: Biochemistry and Biophysics Reports

Article Title: HMGB1 impairs nasal mucosa epithelial barrier function in allergic rhinitis by promoting BECN1-mediating autophagy

doi: 10.1016/j.bbrep.2025.102351

Figure Lengend Snippet: HMGB1 knockdown attenuates autophagy in IL-4-stimulated nasal mucosal epithelial cells. hNEpiC cells were transfected with sh-NC or sh-HMGB1 plasmids and then treated with 10 ng/ml IL-4 for 48 h to establish an inflammatory microenvironment. (A) Representative Western blots and quantitative analysis of key autophagy-related proteins (BECN1, LC3-I/II, ATG5, and p62) in IL-4-treated hNEpiC cells transfected with sh-NC or sh-HMGB1. Decreased LC3-II/LC3-I ratio, BECN1 and ATG5 levels and increased p62 level indicate impaired autophagy flux upon HMGB1 knockdown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Immunofluorescence staining of LC3B (green) in IL-4-treated hNEpiC cells. Nuclei were stained with DAPI (blue). HMGB1 knockdown reduced LC3B puncta formation, suggesting suppression of autophagosome accumulation. Scale bar: 20 μm (C) Autophagic flux was monitored using the mCherry-GFP-LC3B tandem reporter. Yellow puncta (mCherry + GFP+) represent autophagosomes; red-only puncta (mCherry + GFP−) indicate autolysosomes. HMGB1 knockdown inhibit autolysosomes formation. Scale bar: 20 μm.

Article Snippet: The detailed information of all antibodies used in present study are shown as below: Anti -LC3 (14600-1-AP, 1:1000, Proteintech, China), Anti -p62 (18420-1-AP, 1:50000, Proteintech), Anti -Beclin 1 11306-1-AP, 1:1000, Proteintech), Anti -HMGB1 (10829-1-AP, 1:5000, Proteintech), Anti -ATG5 (10181-1-AP, 1:2000, Proteintech), Anti -ZO-1 (21773-1-AP, 1:5000, Proteintech), Anti -Occludin (27260-1-AP, 1:5000, Proteintech), Anti -Claudin 1 (13050-1-AP, 1:1000, Proteintech), Caspase-1 (22951-1-AP, 1:1000, Proteintech), Caspase-4 (67398-1-lg, 1:1000, Proteintech), TNF-α (17590-1-AP, 1:1000, Proteintech), Anti -GAPDH (Bioss, bam-33033 M, 1:10000), Goat Anti-Rabbit IgG H&L (ab6721, 1:5000, abcam, USA), and Rabbit Anti-Mouse IgG H&L (HRP) (ab6728, 1:5000, abcam).

Techniques: Knockdown, Transfection, Western Blot, Immunofluorescence, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: HMGB1 impairs nasal mucosa epithelial barrier function in allergic rhinitis by promoting BECN1-mediating autophagy

doi: 10.1016/j.bbrep.2025.102351

Figure Lengend Snippet: BECN1 knockdown suppresses HMGB1-induced autophagy in hNEpiC cells . The hNEpiC cells were transfected with sh-NC or sh-BECN1 plasmids and treated with 10 ng/ml IL-4 with or without 2 μg/ml recombinant HMGB1 protein for 48 h to evaluate autophagic activity. (A) Representative Western blots and quantitative analysis of autophagy markers. HMGB1 overexpression promoted autophagy as evidenced by increased LC3-II/LC3-I ratio, BECN1 and ATG5 levels and decreased p62 level, while BECN1 knockdown reversed these effects, confirming its essential role in HMGB1-mediated autophagy. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Immunofluorescence analysis of LC3B puncta formation (green). HMGB1 overexpression enhanced LC3B puncta formation, indicating increased autophagosome accumulation, which was suppressed by BECN1 knockdown. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm (C) Autophagic flux assessment using mCherry-GFP-LC3B reporter. Yellow puncta (mCherry + GFP+) represent autophagosomes while red puncta (mCherry + GFP-) indicate autolysosomes. HMGB1 overexpression increased autolysosomes, whereas BECN1 knockdown predominantly reduced autolysosome formation. Scale bar: 20 μm.

Article Snippet: The detailed information of all antibodies used in present study are shown as below: Anti -LC3 (14600-1-AP, 1:1000, Proteintech, China), Anti -p62 (18420-1-AP, 1:50000, Proteintech), Anti -Beclin 1 11306-1-AP, 1:1000, Proteintech), Anti -HMGB1 (10829-1-AP, 1:5000, Proteintech), Anti -ATG5 (10181-1-AP, 1:2000, Proteintech), Anti -ZO-1 (21773-1-AP, 1:5000, Proteintech), Anti -Occludin (27260-1-AP, 1:5000, Proteintech), Anti -Claudin 1 (13050-1-AP, 1:1000, Proteintech), Caspase-1 (22951-1-AP, 1:1000, Proteintech), Caspase-4 (67398-1-lg, 1:1000, Proteintech), TNF-α (17590-1-AP, 1:1000, Proteintech), Anti -GAPDH (Bioss, bam-33033 M, 1:10000), Goat Anti-Rabbit IgG H&L (ab6721, 1:5000, abcam, USA), and Rabbit Anti-Mouse IgG H&L (HRP) (ab6728, 1:5000, abcam).

Techniques: Knockdown, Transfection, Recombinant, Activity Assay, Western Blot, Over Expression, Immunofluorescence

Journal: Biology

Article Title: Divergent Hepatic and Adipose Tissue Effects of Kupffer Cell Depletion in a Male Rat Model of Metabolic-Associated Steatohepatitis

doi: 10.3390/biology14081058

Figure Lengend Snippet: Gadolinium chloride treatment for the last two weeks of diet depletes Kupffer cells and reduces the production of pro-inflammatory cytokines by sucrose-rich diet (SRD). mRNA levels of ( A ) CD68 and ( B ) Clec4f assessed by qPCR. ( C ) Immunofluorescence staining was performed and observed at 400× magnification. Clec4f = red, DAPI = blue. White bar = 50 µm. mRNA levels of ( D ) pro-inflammatory markers and ( E ) anti-inflammatory markers assessed by qPCR. Data is shown as mean ± SEM of n = 8 per group. Statistical significance was obtained by one-way ANOVA followed by Tukey’s post hoc test; * p < 0.05, *** p < 0.001 vs. control; ## p < 0.01 and ### p < 0.001 vs. SRD group.

Article Snippet: The antibodies used in this study were as follows: CLEC4f 1:20, AF2784, and 3-nitrotyrosine (1:50, MAB3248) (R&D systems, Minneapolis, MN, USA).

Techniques: Immunofluorescence, Staining, Control

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 2 ｜ The modified dual SMAD pathway inhibition protocol yielded a low proportion of dopaminergic neurons. (A) Representative images of human induced pluripotent stem cells (hiPSC) with immunostaining of pluripotency markers, octamer-binding transcription factor 4 (OCT4) and stage-specific embryonic antigen-4 (SSEA4). Scale bars: 50 μm. (B) Representative images of neural precursor cells (NPC) induced from hiPSC with immunostaining of SRY-box transcription factor 2 (SOX2) and Nestin. Scale bars: 50 μm. (C) Representative images of hiPSC-derived neurons with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (D) Analysis of neuronal differentiation efficiency (number of cells expressing HuNu: n = 443 cells in dual SMAD pathway inhibition protocol). (E) Representative images of hiPSC-derived neurons with immunostaining of markers for different neuronal subtypes: tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), vesicular glutamate transporter 1 (VGLUT1) (all in green), and MAP2 (red) grown on mouse astrocytes. Scale bars: 50 μm. (F) Differentiation ratio of dopaminergic neurons, GABAergic neurons, and glutamatergic neurons were assessed based on the proportion of TH, GABA, and VGLUT1 positive cells among MAP2 positive cells, respectively. At least three independent replicates were performed of each experiment. SMAD: Suppressor of mother against decapentaplegic.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Modification, Inhibition, Immunostaining, Binding Assay, Derivative Assay, Expressing

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 4 ｜ The SHH + FGF8 differentiation protocol yielded a high proportion of dopaminergic neurons, with a small fraction of neurons that expressed FOXA2, a marker of midbrain dopaminergic neurons. (A) Representative images of neural precursor cells (NPC) induced from human induced pluripotent stem cells (hiPSC) with immunostaining of Forkhead box protein A2 (FOXA2) and SRY-box transcription factor 2 (SOX2). Scale bars: 50 μm. (B) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (C) Comparison of the differentiation efficiency of neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol (number of cells expressing HuNu: n = 607 cells in SHH+FGF8 protocol, n = 443 cells in dual SMAD pathway inhibition protocol). (D) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of tyrosine hydroxylase (TH) (green) and MAP2 (red). Scale bars: 50 μm. (E) The proportion of dopaminergic neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol was determined by calculating the percentage of TH-positive cells in MAP2-positive cells. (number of cells expressing MAP2: n = 581 cells in SHH + FGF8 protocol, n = 339 cells in dual SMAD pathway inhibition protocol). (F) The white box area in the image is enlarged and displayed on the right. NPC were infected with a DAT-Cherry lentivirus and then cultured on a glial cell feeder layer to label dopaminergic neurons in the later differentiation stage. Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of TH (green) and beta III tubulin (TUJ1) (white) on day 33. Scale bars: 50 μm (left), 10 μm (right). (G) The white box area in the image is enlarged and displayed at the bottom. Immunofluorescence staining of FOXA2 (red) and TH (green). Scale bars: 20 μm (upper panels), 5 μm (lower panels). At least three independent replicates were performed for each experiment. Data are presented as mean ± SEM, ****P < 0.0001. Statistical significance was evaluated by two-tailed unpaired Student’s t-test (C, E). FGF8: Fibroblast growth factor 8; ns: not significant; SHH: Sonic Hedgehog; TH: tyrosine hydroxylase.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Marker, Immunostaining, Derivative Assay, Comparison, Modification, Inhibition, Expressing, Infection, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 1. miR-206 targets cyclin D1. (A) Sequence alignment between miR-206 and the 3′UTRs of cyclin D1 from different species. In brackets the 3′UTR size. (B) Diagram of the luciferase reporter construct with the putative miR-206 binding site (WT 3′UTR) and mutations (3′UTR MUT). (C) Relative lucif- erase activity was measured in HeLa cells after transfection of reporter constructs along with pSP65-U1 (CTR) or pSP65–206 (miR-206). Relative Firefly luciferase values were determined by a ratio of Firefly to Renilla luciferase with the control set to 1.00. Values are the means ± SD of 3 separate experi- ments. **A Student t test performed between control and miR-206 transfected cells yielded P values < 0.01.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Sequencing, Luciferase, Construct, Binding Assay, Activity Assay, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 2. Expression kinetics of miR-206 and cyclin D1 in differentiating C2C12 cells. C2C12 myoblasts were seeded in GM at 1.5 × 104/cm2. Cells were shifted in DM 24 h after plating and left to differentiate for further 72 h. (A) Northern blot analysis of miR-206 expression in C2C12 cells after 24 h in GM (0) and at different time points upon shift to DM. (B) Western blot analysis of cyclin D1 and MyHC expression in C2C12 cells cultured as in (A). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) MyHC immunofluorescence staining (green) of C2C12 cells after 24 h in GM (DM 0 h) and after 72 h in DM (DM 72 h). Nuclei were counterstained in blue (DAPI) and individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 20 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Western Blot, Cell Culture, Immunofluorescence, Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 3. miR-206 controls cyclin D1 accumulation in C2C12 cells. C2C12 myoblasts were seeded in GM at 2.5 × 103/cm2. Cells were transfected 24 h after plaiting. (A) Northern blot analysis of miR-206 expression (upper) and western blot analysis of cyclin D1 expression (lower) in C2C12 cells 48 h after transfection with a control vector (CTR) or with a miR-206 expression vector (miR-206). Cells were kept in GM throughout the experiment. (B) The effect of miR-206 overexpression on C2C12 cell proliferation and differentiation was evaluated 48 h after transfection by 1 h BrdU incorporation and MyHC staining, respectively. Results are represented relative to the BrdU+ nuclei or nuclei in MyHC+ cells in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. (C) Immunofluorescence staining of cyclin D1 (pink) and MyHC (green) 48 h after transfection. Nuclei were counterstained in blue with DAPI. Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. To obtain cyclin D1 images, before merging, individual pictures were pseudocol- ored using a LEICA Microsystems Imaging software. Bar = 10 μm. (D) C2C12 myoblasts were seeded at low (LD) and high (HD) density in GM. Cells were shifted to DM the day after plating and analyzed after further 3 d. The panels show a northern blot analysis of miR-206 expression (left panel) and a western blot analysis of cyclin D1 and differentiation associ- ated marker expression (right panel) after 24 h in GM and 72 h after shift- ing to DM. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Northern Blot, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, BrdU Incorporation Assay, Staining, Immunofluorescence, Imaging, Software, Marker

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 4. Inhibition of miR-206 rescues cyclin D1 in myotubes (A) Experimental scheme. C2C12 myoblasts were induced to differ- entiate in DM in the presence of AraC. After 3 d, AraC was washed out and cells left to recover in DM for further 24 h. Finally, pure myotubes were transfected with LNA against miR-206 and analyzed 48 h later. (B) Northern blot analysis of miR-206 and miR-1 expression (left panel) and western blot analysis of cyclin D1 expression (right panel) in pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Cyclin D1 expression in proliferating myoblasts is also shown (GM). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) Double immunofluorescence staining of MyHC and cyclin D1 of pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 10 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Inhibition, Transfection, Northern Blot, Expressing, Western Blot, Control, Double Immunofluorescence Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 5. miR-206 inhibits cell proliferation in Ras-transformed fibro- blasts. (A) Expression levels of cyclin D1 in NIH3T3(Ras) cells as compared with NIH3T3(BN) cells. (B) Real-time PCR analysis of miR-206 expres- sion in NIH3T3(Ras) cells. Results are shown relative to untransformed NIH3T3(BN) cells set to value 1.00. Each sample was analyzed in tripli- cate, and values are the means ± SD of 3 independent experiments. **A Student t test performed between untransformed and transformed cells yielded P values < 0.01. (C) NIH3T3(Ras) cells were transfected with a control vector (CTR) or with a miR-206 expression vector (miR-206) and analyzed 24 h later. Upper, northern blot analysis of miR-206 expression; lower, western blot analysis of cyclin D1 expression. (D) Effect of miR-206 forced expression on cell proliferation as determined by 1 h BrdU incor- poration. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. Equal RNA and protein loading was confirmed by detecting, snRNA U2, and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 6. Relationship between miR-206 downregulation and cyclin D1 expression in NSCLCs. (A) Northern blot analysis of miR-206 in different murine tissues. snRNA U2 levels were used as a loading control. (B) Real-time PCR analysis of miR-206 expression in human NSCLC tissues. Results are shown relative to the matched normal lung tissues set to value 1.00. Each sample was analyzed in triplicate, and values are the means ± SD of three independent experiments. **A Student t test performed between normal and tumor tissues yielded P values < 0.01. (C) Western blot analysis of cyclin D1 expression in normal and neoplastic lung tissues. Equal protein loading was confirmed by detecting actin. n, normal tissue; t = tumor tissue

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 7. miR-206 inhibits cancer cell proliferation through repression of cyclin D1. (A) A549 and HeLa cells were transfected with a control vec- tor (CTR) or with a miR-206 expression vector (miR-206) and analyzed 72 h later. Top panel: northern blot analysis of miR-206 expression; lower panel, western blot analysis of cyclin D1 expression. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (B) Effect of miR-206 forced expression on cell prolifera- tion as determined by 1 h BrdU incorporation and immunofluorescence staining. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values <0.05.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Northern Blot, Western Blot, BrdU Incorporation Assay, Immunofluorescence, Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Pioglitazone inhibits HIF-1α-dependent angiogenesis in rats by paracrine and direct effects on endothelial cells

doi: 10.1007/s00109-013-1115-0

Figure Lengend Snippet: Pioglitazone is associated with reduced gastrocnemius lectin perfusion in vivo. a Representative confocal images of the gastrocnemius muscle of vehicle- ( left ) and pioglitazone-treated rats perfused with lectin ( green ) immediately prior to sacrifice. Pioglitazone decreases the lectin perfusion ( left ) in both diabetic and non-diabetic models compared to vehicle controls. Values are represented as mean ± SEM of the ischemic reserve (the ratio of the signal in the ligated/non-ligated leg) (* p < 0.05 vs. respective placebo-treated control group). Bar 200 μm. b Western blot showing reduced HIF-1α expression in both non-ligated and ligated limbs exposed to pioglitazone (* p < 0.05 vs. vehicle-treated group). Three different blots, each with samples from three different rats, were quantified. Each lane represents muscle tissue from one animal. c Western blot showing reduced VEGF expression in the gastrocnemius muscles with ligated vessels in pioglitazone-treated animals compared to vehicle-treated animals (* p < 0.05 vs. vehicle-treated group)

Article Snippet: Primary antibodies used include VEGF (1:100; Santa Cruz Biotechnologies), HIF-1a (1:100; Abcam, San Francisco, CA), vWF (1:200; Abcam, San Francisco, CA, San Francisco, CA), SMA (1:100; Abcam, San Francisco, CA).

Techniques: In Vivo, Control, Western Blot, Expressing, Muscles

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Pioglitazone inhibits HIF-1α-dependent angiogenesis in rats by paracrine and direct effects on endothelial cells

doi: 10.1007/s00109-013-1115-0

Figure Lengend Snippet: Pioglitazone inhibits HIF-1α in hSkMCs. a Representative immunofluorescence confocal images of hSkMCs in normoxic, hypoxic, or hypoxic plus pioglitazone conditions and stained with HIF-1α ( red ), VEGF ( green ), and the nuclear stain DAPI ( blue ). Pioglitazone reduces nuclear (active) HIF-1α and VEGF expression in hypoxic hSkMCs (* p < 0.05 vs. hypoxia vehicle). Bar 20 μm. b mRNA expression of HIF-1α target genes, HIF-1α and VEGF-R, in hSkMCs exposed to hypoxia plus vehicle or pioglitazone. Pioglitazone reduces HIF-1α and VEGF-R mRNA ( n = 3 experiments. * p < 0.05 vs. hypoxia vehicle)

Article Snippet: Primary antibodies used include VEGF (1:100; Santa Cruz Biotechnologies), HIF-1a (1:100; Abcam, San Francisco, CA), vWF (1:200; Abcam, San Francisco, CA, San Francisco, CA), SMA (1:100; Abcam, San Francisco, CA).

Techniques: Immunofluorescence, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-1β Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin *

doi: 10.1074/jbc.M115.680694

Figure Lengend Snippet: Calmodulin inhibition has no effect on the assembly of the inflammasome or caspase-1 activation. THP-1 cells (10 6 cells/ml) were incubated with LPS (1 μg/ml) for 4 h, with the final 30 min in the presence or absence of E6 berbamine ( E6 berb , 10 μ m , A and B ). These cells were then incubated for another hour in the presence or absence of nigericin ( nig , 10 μ m ). Cells were then fixed and analyzed for ASC expression by immunofluorescence staining, with representative images taken. The number of speck-containing cells was quantified as a percentage of the total number of cells ( n = 3, B ). In addition, 10 7 THP-1 cells (10 6 cells/ml) were incubated with medium or primed with LPS (1 μg/ml) for 4 h, with the final 15 min in the presence or absence of BAPTA-AM (1, 10, or 100 μ m ; C ) or the final 30 min in the presence or absence of E6 berbamine (1 or 10 μ m , D ). Supernatants were then harvested and analyzed by Western blotting ( WB ) using an anti-caspase-1 antibody. A protein marker lane on each gel was used to determine molecular weight. The blots were cropped in each case. Scale bar = 50 μm.

Article Snippet: For immunofluorescence analysis, the primary antibodies used were a rabbit anti-ASC antibody (Santa Cruz Biotechnology), a rabbit anti-calmodulin antibody (Abcam, Cambridge, UK), or a goat anti-human IL-1β antibody (R&D Systems).

Techniques: Inhibition, Activation Assay, Incubation, Expressing, Immunofluorescence, Staining, Western Blot, Marker, Molecular Weight