Journal: Kidney international

Article Title: The L1 cell adhesion molecule is a potential biomarker of human distal nephron injury in acute tubular necrosis.

doi: 10.1038/sj.ki.5002640

Figure Lengend Snippet: Figure 4 | Comparative localization of L1 intra- and extracytoplasmic domains. (a) L1 scheme showing the extracellular part with six immunoglobulin-like domains (Ig-like) (including one recognized by mAb272) and five fibronectin III-like (FNIII) domains followed by the transmembrane segment and cytoplasmic fragments (recognized by the goat polyclonal serum). (b–d) Normal CDs are compared with (e–g) damaged CDs. (b, e) CDs are stained by goat polyclonal antiserum labeled by FITC (green) and (c, f) mAb272 labeled by rhodamine (red). (d, g) Merge. (d) In normal CD, both antibodies give a basolateral signal. (g) In injured principal cells, the basolateral signal is low with both antibodies, whereas there is a strong apical membrane (arrow) signal with the anti-cytoplasmic domain antibody contrasting with no signal for the extracellular fragment. Original magnification 630.

Article Snippet: After four washes with TBS-T (Tris-buffered saline with 0.1% Tween), goat anti-human L1 extracellular domain antibody was added (R&D Systems), followed by anti-goat coupled to alkaline phosphatase (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Staining, Labeling, Membrane

Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a, Top: Scheme of metabolomic analysis using extracts from nerve segments. Bottom: Concentrations of key energy metabolism intermediates in control and axotomized nerve segments from C57Bl/6J mice (F6P/G6P: fructose-6-phosphate/glucose-6-phosphate. FBP: fructose-1,6-bisphosphate. GI-OH3P: Glyceraldehyde-3-phosphate. 2PG/3PG: 2-phosphoglycerate/3-phosphoglycerate. LACT: lactate) (Error bars represent s.e.m. n=5 mice per condition and metabolite). b, Top: Scheme of extracellular flux analysis of SCs purified from C57Bl/6J mouse nerves. Bottom: Box and whiskers plot (maximum, 25th percentile, median, 75th percentile, minimum) shows glycolytic activity parameters as assessed by extracellular acidification rate (ECAR) measurements in control mouse SCs and cells with Nrg1-induced ErbB2 activation (n=9 well preparations per condition, *P=0.0080, **P=0.0464, ***P<0.0001). c, Western blot analysis (cropped blot images) of control- and Nrg1-treated C57Bl/6J mouse SCs probed with the indicated antibodies. (n=6 independent pair preparations (2 separate dishes) for PFKFB3, and n=3 independent pair preparations (2 separate dishes) for LDHA quantification). d, Intracellular and extracellular (supernatant) lactate concentrations from control and Nrg1-treated mouse SC preparations normalized to cell number and cellular protein. Note decreased intracellular and increased extracellular lactate levels in SCs treated with Nrg1 for 24h, indicating greatly increased lactate extrusion (Error bars represent s.e.m. n=3 well preparations from 3 independent experiments per condition).

Article Snippet: SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures.

Techniques: Control, Purification, Activity Assay, Activation Assay, Western Blot

Journal: European journal of pharmacology

Article Title: Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence.

doi: 10.1016/j.ejphar.2024.176397

Figure Lengend Snippet: Fig. 3. TZ reduces AAA formation by attenuating PEG3 signaling. (A) Heatmap depicting differentially expressed genes between control and 10 nM TZ-treated MOVAS cells. (B) Representative images of IHC staining for PEG3 in human AAA samples (scale bar, 50 μm) and quantification of IHC results (n = 3). Black arrows indicate PEG3-positive cells. *p < 0.05 compared with the control group. (C) MOVAS cells were treated by 1 μM Ang II with or without TZ for 48 h. Representative western blots and quantification of PEG3 and MMP2 protein expressions in MOVAS (n = 3–6). *p < 0.05 compared with ctrl group, #p < 0.05 between Ang-TZ and Ang II group. (D) (Upper) Representative IHC staining images of PEG3 in Apoe−/−mice suprarenal aortas from each group (scale bar, 50 μm) and quantification of IHC results (n = 5–9). *p < 0.05 compared with the sham-water group, #p < 0.05 compared with the Ang II-water group. (Lower) Representative IHC staining images of PEG3 in C57BL/6J mice infrarenal aortas from each group (scale bar, 50 μm) and quantification of IHC results (n = 5–12). *p < 0.05 compared with the sham-water group, #p < 0.05 compared with the CaCl2-water group. Black arrows indicate PEG3-positive cells. (E) (Left) Representative Immunofluorescent staining images of PEG3 (Green), α-SMA (Red) and DAPI (Blue) in Apoe−/−mice suprarenal aortas from sham-water group, Ang II-water group elastin unbroken area, Ang II- water group elastin broken area and Ang II-TZ (100 μg//kg) group (scale bar, 50 μm). (Right) Representative Immunofluorescent staining images of PEG3 (Green), α-SMA (Red) and DAPI (Blue) in C57BL/6J mice infrarenal aortas from the indicated groups (scale bar, 50 μm). IHC, Immunohistochemistry. α-SMA, α-smooth muscle actin.

Article Snippet: Primary antibodies used included: PEG3 (AF9152, 1:150, Affinity, China), α-SMA (BM0002, 1:200, BOSTER, Wuhan, China), p21 (sc-6246, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (10375-2-AP, 1:300, Proteintech, Chicago, IL, USA), MMP2 (10373-2-AP, 1:400, Proteintech), and F4/80 (#70076, 1:200, CST, Boston, MA, USA).

Techniques: Control, Immunohistochemistry, Western Blot, Staining

Journal: European journal of pharmacology

Article Title: Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence.

doi: 10.1016/j.ejphar.2024.176397

Figure Lengend Snippet: Fig. 6. Silencing Peg3 alleviates Ang II-induced cell senescence and apoptosis in MOVAS. MOVAS cells were exposed to 1 μM Ang II with or without Peg3 knockdown for 48 h. (A) Representative western blots and (B) quantification of PEG3, MMP2, p53, cleaved caspase-3 p21, p16 and p-H2AX protein expressions (n = 3). (C) Relative mRNA levels of Peg3 analyzed by RT-qPCR (n = 6). (D) Relative mRNA levels of Mmp2, Mmp9, p53, p21 and p16 analyzed by RT-qPCR (n = 6). (E) (Left) Representative flow-cytometric plots of Annexin V-APC/7-AAD staining. (Right) Quantification of early apoptosis (Annexin V(+)/7-AAD(−)) and total apoptosis (Annexin V(+)) percentages (n = 6). (F) (Left) Representative images of SA-β-gal staining in MOVAS (scale bar, 50 μm) and (Right) quantification of SA- β-gal positive cells (n = 3). (E) Relative mRNA levels of SASP factors including Il-6, Il-1β, Ccl2, Ccl7, Cxcl1, Cxcl10 and Cxcl12 analyzed by RT-qPCR (n = 3). *p < 0.05 (si-Peg3 group vs NC group) or (si-Peg3+Ang II group vs NC + Ang II group), #p < 0.05 (NC + Ang II group vs NC group) or (si-Peg3+Ang II group vs si-Peg3 group).

Article Snippet: Primary antibodies used included: PEG3 (AF9152, 1:150, Affinity, China), α-SMA (BM0002, 1:200, BOSTER, Wuhan, China), p21 (sc-6246, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (10375-2-AP, 1:300, Proteintech, Chicago, IL, USA), MMP2 (10373-2-AP, 1:400, Proteintech), and F4/80 (#70076, 1:200, CST, Boston, MA, USA).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Staining

Journal: European journal of pharmacology

Article Title: Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence.

doi: 10.1016/j.ejphar.2024.176397

Figure Lengend Snippet: Fig. 7. Schematic illustration shows the mechanisms of TZ inhibition on AAA formation. Stress induces the expression of Peg3 in VSMCs, triggering VSMCs senescence, apoptosis, and ECM degradation, eventually leading to AAA formation Treatment with low-dose TZ reduces Peg3 expression, reversing these effects. The picture was drawn by Figdraw. VSMCs, vascular smooth muscle cells. SASP, senescence-associated secretory phenotype. ECM, extracellular matrix.

Article Snippet: Primary antibodies used included: PEG3 (AF9152, 1:150, Affinity, China), α-SMA (BM0002, 1:200, BOSTER, Wuhan, China), p21 (sc-6246, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (10375-2-AP, 1:300, Proteintech, Chicago, IL, USA), MMP2 (10373-2-AP, 1:400, Proteintech), and F4/80 (#70076, 1:200, CST, Boston, MA, USA).

Techniques: Inhibition, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 4. Effects of CAR agonists on CFTR-mediated Cl−

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 5. Effects of CAR agonist on mRNA and protein expres- sion of CFTR in T84 cells (A): Quantitative real time PCR ana- lysis of CFTR mRNA expression in T84 cells after treatment with vehicle (0.01% DMSO), CITCO, or phenytoin at the indicated concentrations for 24 h. The results were normalized to GAPDH. (B) Representative blot and densitometric quantification of CFTR protein expression were normalized to β-actin levels in T84 cells treated with vehicle (0.01% DMSO), CITCO, or phenytoin at the indicated concentrations for 24 h. The data are expressed as means ± S.E. (n = 5–7). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to control.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Lysis, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 6. Effects of CAR antagonist on CITCO- induced inhibition of Cl−secretion and CFTR protein expression in T84 cells (A). Effects of CAR antagonist on CITCO-induced inhibition of Cl−secretion in T84 cells. T84 cells were treated with vehicle (0.1% DMSO), CITCO (1 μM), CAR antagonist [CINPA1 (1 μM)], or CITCO plus CINPA1 for 24 h, then the forskolin-stimulated Isc was measured. (left) Representative Isc tracings and (right) summary of the data are shown. Data are ex- pressed as means ± S.E. of percentages of forskolin-stimulated Isc (n = 5). (B) Representative blot and densitometric quanti- fication of CFTR protein expression in T84 cells treated with vehicle alone or together with CITCO (1 μM) and/or CINPA1 (1 μM) for 24 h. The data are expressed as means ± S.E. (n = 5). *, P < 0.05 compared to the vehicle- treated control. NS, not significantly different from control. ##, P < 0.05; ##, P < 0.01 compared to CITCO-treatment group.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Inhibition, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 7. Effect of CAR activation on CFTR-mediated Cl−transport during inhibition of mRNA synthesis in T84 cells. T84 cells were treated with ve- hicle (0.1% DMSO), CITCO (1 μM), or actinomycin D (Act D) (5 μg/ml), or CITCO plus Act D for 24 h, after that CFTR expression was measured by real- time PCR analysis. The results were normalized to GAPDH. The data are ex- pressed as means ± S.E. (n = 5). *, P < 0.05; ***, P < 0.001 compared to the vehicle-treated control. NS, not significantly different.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Activation Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

doi: 10.1016/j.biopha.2019.01.015

Figure Lengend Snippet: Fig. 9. Effect of CAR activation on CFTR expression and function in mouse intestine. (A): Activation of CAR target gene (MDR1) in mouse intestine. B: Quantitative PCR analysis of CFTR mRNA expression in mouse intestinal tissues after intraperitoneally injected the mice with 3 mg/kgBW TCPOBOP (CAR agonist) or vehicle (corn oil) for 7 days. The results were normalized to GAPDH. C: Representative blot and densitometric quantification of CFTR pro- tein expression in mouse intestinal tissues after treated the mice with TCPOBOP or vehicle (corn oil) for 7 days. The results were nor- malized to β-actin levels. The data are pre- sented as mean ± SE (n = 8). *, P < 0.05; **, P < 0.01 compared to control. D. Antidiarrheal application of CAR activation on cholera toxin-induced intestinal fluid accumu- lation in mouse closed-loop model. ICR mice were treated with 3 mg/kgBW TCPOBOP or vehicle (corn oil) for 7 days. After treatment, PBS or PBS containing cholera toxin (CT) (1 μg/loop) were instilled into ileal loops. Four hours later, ileal loops were removed for de- termination of loop weight/length ratio. Representative photograph of ileal loops and summary of the data are shown. Data are ex- pressed as means ± S.E. of loop weight/ length ratio (n = 8). **, P < 0.01 compared with CT-treated control. NS, not significantly different compared with PBS-treated control.

Article Snippet: After centrifugation of the cell and tissue homogenates at 12,000 rpm for 20min at 4 °C, equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK).The membrane was then incubated for 1 h with 5% non-fat milk and probed overnight at 4 °C with anti-human CFTR, anti-β actin (Cell signaling), anti-mouse CFTR (Abcam) and anti-CAR (R&D systems) (all at 1000-fold dilution) followed by a further incubation with HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Injection, Control