Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: CYP27C1 expression in different human lung cancer cell lines. ( A ) Relative mRNA expression levels of CYP27C1 in human bronchial epithelial BEAS-2B cells, and human lung cancer cells were detected by real-time fluorescence quantitative PCR. Results comparing BEAS-2B with other cell lines were analyzed by unpaired two-tailed T test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., p > 0.05. ( B , C ) Protein expression level of CYP27C1 in BEAS-2B, and human lung cancer cells were detected by immunoblot analysis. Grey scales of the bands were analyzed by Gel-Pro ANALYZER. Results were analyzed by unpaired two-tailed T test comparing BEAS-2B with other cell lines. *, p < 0.05; **, p < 0.01; n.s., p > 0.05.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Expressing, Fluorescence, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: Construction of stable CYP27C1-knockdown or stable CYP27C1-overexpressed human lung cancer cell lines. ( A ) Protein level of stable CYP27C1-knockdown A549, H1975 cells, stable CYP27C1-overexpressd H460 cells, and the corresponding non-transfected control and vector control cells was detected by immunoblot analysis. ( B ) Grey scales of the bands were analyzed by Gel-Pro ANALYZER. Results were analyzed by unpaired two-tailed T test. Lines with blunt arrow indicated the comparison between different groups. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 n.s., p > 0.05.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, Transfection, Control, Plasmid Preparation, Western Blot, Two Tailed Test, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: Stable CYP27C1-knockdown increases basal tyrosine phosphorylation level. Protein level of stable CYP27C1-knockdown A549 ( A ) stable CYP27C1-knockdown H1975 cells ( B ) and the corresponding control cells was detected by immunoblot analysis. Grey scales of the bands were measured by Gel-Pro ANALYZER. Results were analyzed by unpaired two-tailed T test. Lines with blunt arrow indicated the comparison between different groups. *, p < 0.05.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, Phospho-proteomics, Control, Western Blot, Two Tailed Test, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: Stable CYP27C1-knockdown enhances colony formation ability of lung cancer cells. ( A – D ) A549-shCYP27C1, H1975-shCYP27C1, and the corresponding control cells were seeded in 6-well plates, respectively. Culture medium was changed in every 3–4 days, and cell colony formation was observed. After culturing for about 14 days, cell colonies were stained with crystal violet, and scanned. The absorbance of eluent was detected at 595 nm. Results were analyzed by unpaired two-tailed T test. ***, p < 0.001, ****, p < 0.0001.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, Control, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: Stable CYP27C1-knockdown promotes human lung cancer cell proliferation in vitro, and in xenograft mice model. ( A , B ) Cell viability of A549-shCYP27C1, H1975-shCYP27C1, and the corresponding control cells was detected by MTS assay at different time points. Time-cell proliferation curves were plotted, and analyzed by two-way ANOVA. ****, p < 0.0001. ( C ) BALB/c nude mice (N = 10) were divided into two groups. H1975-shCYP27C1 cells were injected into the right armpit of the mice in experimental group, whereas H1975-shCtrl cells were injected into mice in control group. Tumor formed on the ninth day. Length ( l ) and width ( w ) of the tumor was measured on 12, 15, 18, 22, 24, and 26 days after injection. Tumor volume was calculated by formula: V = (l×w 2 )/2 . Time-tumor volume curve was plotted, and analyzed by two-way ANOVA. n.s., p > 0.05. ( D ) Mice were sacrificed on the twenty-seventh day after injection. Mice were dissected, tumor weight was measured, and analyzed with Mann–Whitney test. *, p < 0.05. ( E ) Histology (hematoxylin-eosin staining) of representative xenograft tumor in nude mice model. Magnification: 50× ( panel 1,3 ); 200× ( panel 2,4 ).

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, In Vitro, Control, MTS Assay, Injection, MANN-WHITNEY, Staining

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: CYP27C1 expression affects ability of migration in lung cancer cells. ( A , B ) H1975-shCYP27C1, H460-CYP27C1, and the corresponding control cells were seeded in 12-well plates for 24 h before cell scratch assay. The wound areas were captured at different time points (0 h and 24 h). Scratching wound healing rate was analyzed by ImageJ software. Results were analyzed by unpaired two-tailed T test. *, p < 0.05, **, p < 0.01.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Expressing, Migration, Control, Wound Healing Assay, Software, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: Stable CYP27C1-knockdown alters Akt-p53 signal transduction in human lung cancer cells. ( A – C ) A549-shCYP27C1, H1975-shCYP27C1, and the corresponding control cells were seeded in 6-well plate for 24 h before protein extraction and quantitation. Total Akt, phosphorylated Akt, p53, and β-actin were detected by immunoblot analysis using the corresponding antibodies. Grey scales of the bands were measured by Gel-Pro ANALYZER, and analyzed by unpaired two-tailed T test. *, p < 0.05, n.s., p > 0.05.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, Transduction, Control, Protein Extraction, Quantitation Assay, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: CYP27C1-knockdown attenuates anticancer potency of protein kinase inhibitors. ( A – E ) A549-shCYP27C1, H1975-shCYP27C1, and the corresponding control cells were treated with indicated concentration of protein kinase inhibitors (picropodophyllin, pacritinib, and SKLB610). Real-time cell index reflecting cell viability were detected by RTCA-S16 System. Results were analyzed by two-way ANOVA. ****, p <0.0001, **, p <0.01, *, p < 0.05, n.s., p > 0.05. ( F ) Stable CYP27C1-knockdown H1975 cells and the corresponding control cells were treated with indicated concentrations of SKLB610, cell viability was determined by MTS assay at different time point, and analyzed by two-way ANOVA. ****, p <0.0001.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, Control, Concentration Assay, MTS Assay

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: CYP27C1 impacts inhibitory effect of picropodophyllin via IGF-1R/AKT/p53 signaling pathway. ( A , B ) A549-shCYP27C1, H1975-shCYP27C1, and the corresponding control cells were seeded in 6-well plate for 24 h before treatment of PPP (100 μM, 24 h). Protein samples were applied to perform immunoblot analysis after extraction and quantitation. Total Akt, phosphorylated Akt, p53, and β-actin were detected by immunoblot analysis using the corresponding antibodies. ( C , D ) Grey scales of the bands were measured by Gel-Pro ANALYZER, and analyzed by unpaired two-tailed T test. *, p < 0.05, **, p < 0.01, ***, p < 0.001, n.s., p > 0.05.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Control, Western Blot, Extraction, Quantitation Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway

doi: 10.3390/ijms23147853

Figure Lengend Snippet: CYP27C1-knockdown impairs anticancer effect of vinorelbine. ( A – C ) A549-shCYP27C1, H1975-shCYP27C1, H460-CYP27C1, and the corresponding control cells were seeded in 24-well plates, respectively. After seeding for 24 h, cell medium was changed into that containing different concentrations of vinorelbine (0, 1.25, 2.5, 5, 10, 20 nM or 0, 2.5, 5, 10, 20, 40 nM). After treatment for 72 h, NBB assays were performed to detect cell viability. Results were analyzed by two-way ANOVA. ****, p < 0.0001, ***, p < 0.001.

Article Snippet: As previously described, diluted primary antibodies targeting CYP27C1 (1:800, GTX45438, Gene Tex, Irvine, CA, USA), beta-actin (1:, A5441, Sigma Aldrich, St. Louis, MO, USA), phospho-tyrosine (1:2000, #9416, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (1:1000, #9102, Cell Signaling Technology), phosphorylated ERK1/2 Thr202/Tyr204 (1:2000, #4370, Cell Signaling Technology), Akt (1:1000, #4691, Cell Signaling Technology), phospho-Akt Ser473 (1:1000, #9271, Cell Signaling Technology), and p53 (1: 1000, sc-6243, Santa Cruz Biotechnology) were applied to probe the protein on PVDF membrane at four degrees Celsius overnight, following by the reaction with secondary anti-mouse or anti-rabbit antibodies at room temperature for two hours.

Techniques: Knockdown, Control

Journal: bioRxiv

Article Title: Nonlethal effects of predation: Presence of insectivorous birds affects the behaviour and level of stress in insects

doi: 10.1101/2022.04.01.486535

Figure Lengend Snippet: Effect of predator treatments on the Schgr-AKH-II level in CNS (a) and in haemolymph (b) of S. gregaria within the laboratory experiments. Statistically differences between the groups were evaluated using one-way ANOVA with Tukey’s posttest, and significant results are marked by *** P <0.001, * P <0.05, ns P >0.05. Treatments: Bird = real great tits were present in the experimental cages and warning calls were played, Call = only warning call of great tit was played, Control = only locusts were present in the cages.

Article Snippet: The primary rabbit antibodies used in the procedure (dilution 1:1000) were raised commercially against the Schgr-AKH-II by Moravian-Biotechnology (Brno, Czech Republic); to exclude a possible cross-reactivity, the corresponding pre-immune serum was used as well.

Techniques:

Journal: bioRxiv

Article Title: Nonlethal effects of predation: Presence of insectivorous birds affects the behaviour and level of stress in insects

doi: 10.1101/2022.04.01.486535

Figure Lengend Snippet: Effect of predator treatments on the Schgr-AKH-II level in CNS (a) and in haemolymph (b) of S. gregaria in the outdoor aviary experiments. Statistically significant differences between the groups were evaluated using the Student’s t -test, and significant results are marked by * P < 0.05, ns P > 0.05 ( n = 4-16). Treatments: Bird = real great tits were present in the aviary and warning calls were played, Control = only locusts were present in the nearby shady area without birds.

Article Snippet: The primary rabbit antibodies used in the procedure (dilution 1:1000) were raised commercially against the Schgr-AKH-II by Moravian-Biotechnology (Brno, Czech Republic); to exclude a possible cross-reactivity, the corresponding pre-immune serum was used as well.

Techniques:

Journal: The FASEB Journal

Article Title: Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality

doi: 10.1096/fj.201900246RR

Figure Lengend Snippet: Genetic deletion of Per1/Per2 enhances pulmonary leukocyte infiltration and lung tissue damage, which contribute to an early mortality in SCD. A) Schematic of SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. B) Images of irradiated male mice ∼16 wk post-BMT. C) Increased mortality was identified beginning 42 d and up to 112 d after irradiation and hematopoietic adoptive transfer in female SCD BM transplanted to Per1/Per2 dKO mice. D) Panel of histologic stains, hematoxylin and eosin (H&E), and Ly6-positive leukocytes as indicated with red arrows in SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. E) Semiquantification of histologic assessment of lung isolated from SCD or WT phenotypic mice with or without Per1/Per2. F) Quantification of leukocytes in ×20 field in whole lung sections isolated from SCD and WT BM transplant mice; n = 5 mice/group. ND, not determined. *P < 0.05, SCD → WT compared with WT → WT mice, **P < 0.01, SCD → WT compared with SCD → Per1/Per2 dKO mice.

Article Snippet: Sections were incubated with Ly6 primary antibody (rat monoclonal anti-mouse, 1:500 dilution in blocking solution; BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Irradiation, Adoptive Transfer Assay, Isolation

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: Activation of DR1 upregulates the CSE/H 2 S pathway in the kidneys of T1D mice. (A and C) The expression of DR1 and CSE in the kidneys was determined by western blotting (n=3). The intensity of each band was quantified by densitometry and data was normalized to the β-actin signal. (B and D) H 2 S levels were detected by ultraviolet spectrophotometer in the kidneys (n=6). The results were expressed as the mean ± standard error of the mean. * P<0.05, **P<0.01 vs. control group; ## P<0.01 vs. T1D-12 W group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; T1D, diabetes group; W, week.

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay, Expressing, Western Blot, Spectrophotometry

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: DR1 activation inhibits cell proliferation and collagen deposition by upregulates the CSE/H 2 S pathway in the kidneys of T1D mice. Morphology of kidney by hematoxylin and eosin staining (magnification, ×400; scale bars, 100 µ m), collagen deposition by Masson staining (magnification, ×400; scale bars, 100 µ m, the blue stain by the arrow indicated is collagens) and thickness of renal basement membrane by transmission electron microscope (magnification, ×20,000; scale bars, 2 µ m). Renal injury was aggravated, including glomerular hypertrophy, collagen deposition in interstitial kidney tissue and the thickness of the glomerular basement membrane were increased in the T1D-4 W, T1D-8 W and T1D-12 W groups. These changes were most significant in the T1D-12 W group. Pretreatment with SKF38393 or NaHS significantly reduced these pathological changes in the T1D-12 W group. All data were from at least 4 independent experiments. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; T1D, diabetes group; W, week.

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay, Staining, Transmission Assay, Microscopy

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: DR1 activation increases the CSE/H 2 S pathway in the HG-induced MCs. (A) The expression of DR1 and CSE in the HG-induced MCs was determined by western blotting (n=3). The intensity of each band was quantified by densitometry and data was normalized to the β-actin signal. (B) H 2 S levels were detected by fluorescence microscopy in the HG-induced MCs (n=6, magnification, ×200; scale bars, 200 µ m). The results were expressed as the mean ± standard error of the mean. * P<0.05, ** P<0.01 vs. control group; # P<0.05, ## P<0.01 vs. HG group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell.

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay, Expressing, Western Blot, Fluorescence, Microscopy

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: Activation of the DR1-CSE/H 2 S pathway attenuates HG-stimulated MC proliferation. (A) Cell viability was measured by CCK-8 assay (n=8). (B) Cell proliferation rate was measured by EdU assay (n=6, magnification, ×200; scale bars, 200 µ m). (C) Quantify the results of (B) with a histogram (n=6). (D) The expression of cyclin D1, PCNA and P21 in the HG-induced MCs was determined by western blotting (n=3). The results were expressed as the mean ± standard error of the mean. * P<0.05, ** P<0.01 vs. control group; # P<0.05, ## P<0.01 vs. HG group; & P<0.05, && P<0.01 vs. HG + SKF38393 group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell.

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay, CCK-8 Assay, EdU Assay, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: Activation of the DR1-CSE/H 2 S pathway inhibits HG-induced collagen deposition in MCs. The expression of COL1, α-SMA and MMP9 in the HG-induced MCs was determined by western blotting. The experiments were repeated at least three times. The results were expressed as the mean ± standard error of the mean. * P<0.05, ** P<0.01 vs. control group; # P<0.05, ## P<0.01 vs. HG group; & P<0.05, && P<0.01 vs. HG + SKF38393 group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell; COL1, collagen 1; α-SMA, α-smooth muscle actin.

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: Activation of the DR1-CSE/H 2 S pathway downregulates ERK1/2 pathway in HG-induced MCs. The activity of p-ERK1/2 and t-ERK1/2 was detected through western blotting from different groups in HG-induced MCs. PD98059 is an ERK1/2 inhibitor. The intensity of each phosphorylated band was quantified by densitometry and data was normalized to the corresponding total band signal. The experiments were repeated at least three times. The results were expressed as the mean ± standard error of the mean. * P<0.05 vs. control group; # P<0.05, ## P<0.01 vs. HG group; & P<0.05 vs. HG + SKF38393 group. DR1, dopamine 1 receptors; CSE, cystathionine-γ-lyase; HG, high glucose; MC, mesangial cell; p-, phosphorylated; t-, total;

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay, Activity Assay, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: The DR1-CSE/H 2 S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice

doi: 10.3892/ijmm.2021.5062

Figure Lengend Snippet: The mechanism of DR1 activation inhibiting ECM deposition and MC proliferation. DR1 activation increases (Ca 2+ ) i and then enhances interaction of CaM and CSE and promotes generation of endogenous H 2 S, which inhibits MC proliferation and ECM deposition by downregulating ERK1/2 signaling pathway. DR1, dopamine 1 receptors; ECM, extracellular matrix; MC, mesangial cell; CaM, calmodulin; CSE, cystathionine-γ-lyase; p-, phosphorylated.

Article Snippet: The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Experimental exposure to Lutzomyia longiplapis sand flies induces HO-1 and Nrf2 protein expression at bite sites in healthy human volunteers. (A,B,D,E) Immunohistochemistry of paraffin-embedded sections of skin specimens obtained by biopsy of the bite sites 48 h after sand fly exposure. (A) HO-1 staining in sand fly-exposed skin, 400X. (B) Higher magnification from image (A) (1000X). (C) Nrf-2 staining in sand fly-exposed skin, 400X. (D) Higher magnification from image (C) (1000X). (E) HO-1 staining in unbitten skin (1000X). (F) Nrf-2 staining in unbitten skin (1000X). Bar, 20 μm. Red arrows point to cells positively stained with HO-1 or Nrf2. Digital images 400 × (lower magnification) or 1000X (higher magnification) were captured using a Nikon E600 microscope and an Olympus Q-Color 1 digital camera with the Image Pro Plus software. (G) Plasma HO-1 levels in individuals ( n = 7) before and after nine exposures to sand fly bites were measured by ELISA. Plasma samples were collected 48 h after each exposure to sand fly bites. Each line represents one individual volunteer. Data represent individual values and were compared using the Kruskal-Wallis test ( p = 0.08).

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Expressing, Immunohistochemistry, Staining, Microscopy, Software, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Bites of Lutzomyia longipalpis sand flies induce Nfr2-dependent HO-1 protein expression by resident macrophages in mice. Ears of C57BL/6 mice were exposed to bites of 20 uninfected L. longipalpis sand flies (UB, uinfected bite) and tissue was processed for total protein extraction before or after sorting by flow cytometry. (A,B) Western blots of total protein extracts from mice ears. Hsp90 was used as a loading control. (A) kinetics of HO-1 expression following sand fly bites. Total protein extract from a mouse spleen was used as a positive control. (B) HO-1 expression in wild-type, Nrf2 knockout (Nrf2 −/− ) and IL-10 knockout (IL-10 −/− ) mice 24 h after exposure to sand fly bites. (C,D) Ear cells recovered 24 h after sand fly bites were stained with antibodies for population sorting by flow cytometry prior to total protein extraction. (C) Gating strategy for sorting populations of non-myeloid cells (I), neutrophils (II), resident macrophages (III) and inflammatory monocytes (IV). (D) Western blot of total protein extract from populations sorted according to (C) . Naïve total ear cells were used as a negative control (CT). Histone H3 was used as a loading control. Blots are representative of two to three independent experiments.

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Expressing, Protein Extraction, Flow Cytometry, Western Blot, Positive Control, Knock-Out, Staining, Negative Control

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Lutzomyia longipalpis saliva activates ARE, nuclear translocation of Nrf2, and HO-1 expression in human macrophages. (A) THP-1 derived macrophages were plated on coverslips and stimulated with either the equivalent of one pair of salivary gland sonicate (SGS) per well or 30 μM CoPP (cobalt protoporphyrin IX) for 24 h, then stained for HO-1. Cells were counter-stained with 1 μg/mL of DAPI to visualize the nucleus. Orange and Blue colors indicate positive staining for HO-1 and nuclei, respectively. Bar, 20 μm. Digital images (400 × original magnification) were captured on a Nikon E600 microscope coupled to an Olympus Q-Color 1 digital camera, and visualized using Image Pro Plus software. (B,C) THP-1 macrophages were treated with one pair SGS per well, followed by extraction of total protein, and detection by Western blot. (B) HO-1. (C) Nrf-2. β-actin and Lamin B were used as loading controls. (D) RAW 264.7 cells were transiently transfected with p3xARE- or pNrf2-promoterluciferase reporter plasmids constructs and treated with 30 μm CoPP or 10 mM DL-sulforaphane (SFN) or one pair SGS per well for 24 h post-transfection. Whole-cell lysates were analyzed for luciferase activity of p3xARE and Nrf2. Data are representative of three independent experiments.

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Translocation Assay, Expressing, Derivative Assay, Staining, Microscopy, Software, Western Blot, Transfection, Construct, Luciferase, Activity Assay

Journal: Frontiers in Immunology

Article Title: Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

doi: 10.3389/fimmu.2018.02779

Figure Lengend Snippet: Lutzomyia longipalpis saliva induces HO-1 protein expression at bite sites in mice. C57BL/6 mice were injected intradermally in the ear with the equivalent of 1 pair of L. longipalpis salivary gland sonicate (SGS). (A) Western blot of whole-tissue extract 24 h after sand fly exposure. β-actin was used as a loading control. (B) Optical density of the bands in (A) was quantified using ImageJ software. Data are presented as the ratio of HO-1 to β-actin. Data are representative of two independent experiments.

Article Snippet: For immunohistochemistry, primary antibodies (diluted at 1:100) against HO-1 (Enzo, ADI-SPA-896-J, Ann Harbor, MI) and Nrf2 (Abcam, ab31163, Cambridge, MA) were used.

Techniques: Expressing, Injection, Western Blot, Software