Annexin V-FITC/PI细胞凋亡检测试剂盒

The apoptotic rate of cells. IBV Beaudette-infected cells (10 MOI) were subjected to flow cytometry at different times. Data are shown as the mean SEM of three independent experiments. * p < 0.05,** p < 0.01 versus control group (0 h).

Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. *P < 0.05 vs. normal group (three replicates)

Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24,and 48 h during reoxygenation after OGD when compared with normal astrocytes. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surroundingmedium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates)

a. Protein expression of BMSCs in Col/HA and HA hydrogels determined by western blotting after osteogenic induction for 2 weeks. b. Semi-quantitative analysis of protein expression levels normalized to beta-actin. n =3,***p < 0.001

Effects of different addition time of CaCl2 (a) and different addition concentrationsofCaCl2(b)onDCW,curvulamineproduction,andYp/xat144h (180rpm, 28°C) by Curvularia sp. IFB-Z10. * Indicates statistical significance (P < 0.05)comparedtothecontrolwithoutCaCl2 addition.

Effects of VRP andCaCl2 on intracellularCa2+ level (a), andextracellular Ca2+ level (b). * Indicates statistical significance(P < 0.05) compared to the control without VRP and CaCl2 addition. Comparison between the cytosolic Ca2+ images of Curvularia sp. observed by light microscopy (c1, c3, c5, c7) and fluorescencemicroscopy(c2,c4,c6,c8)(×20)at48h.Control(c1,c2);5mMCaCl2 (c3,c4);10μMVRP(c5,c6);10μMVRPplus5mMCaCl2 (c7,c8).

Comparisonbetweenthekineticprofilesofcurvulamineproduction(a), residualsugar(b), meandiameter ofpellet (c),andnumberof pellets per mL broth (d) under control and 5mM CaCl2 addition. * Indicates statistical significance (P < 0.05) compared to the control without CaCl2 addition. ComparisonbetweenthesurfacemorphologyofCurvulariasp.underscanning electronmicroscopy(e).0h×250(e0).Control48h×100(e1);5mMCaCl2 48h×100 (e2). Control 144h×100 (e3); 5mM CaCl2 144h×100 (e4). ComparisonbetweenthepelletinternalstructureofCurvulariasp.observedby lightmicroscopy(f).Control144h×4(f1);5mMCaCl2 144h×4(f2).

Response of transcriptional level of spt1 (a), spt2 (b), and aons (c) to CaCl2 addition.*Indicatesstatisticalsignificance(P < 0.05)comparedtothe controlwithoutCaCl2 addition.

ResponseofexpressionlevelofSPT1(a)andSPT2(b)toCaCl2addition. *Indicatesstatisticalsignificance(P < 0.05)comparedtothecontrolwithout CaCl2 addition.

Time courses of DO, DCW, curvulamine production, pH, and residual sugar in Curvularia sp. fermentation process with CaCl2 addition in 5-L bioreactor(agitationspeedat250rpm,aerationrateat1.0VVM,and28°C).

Upregulation of taurine transporter in adipogenic differentiation. A. Transcriptome sequencing of hASCs treated with or without adipogenic inducer. Gene expression profiles were performed with log10 (RPKM+1) value, with high expression gene in blue and low expression gene in red. Color from red to blue indicates higher gene expression. B. The mRNA expression of TauT in the presence or absence of inducer. hASCs were induced by the adipogenic components for 3 days and mRNA levels were analysed by quantitative PCR and normalised to β-actin (n=3, *p<0.05). C. The protein expression of TauT in the presence or absence of inducer. hASCs were induced by the adipogenic components for 3 days. FABP4 were detected as adipogenic differentiation marker. D. The mRNA levels of TauT in subcutaneous fat tissues of ob/ob or db/db mice. Mice at the age of 12 weeks and age-matched wild type were excised and extracted for RNA (n=5, ***p<0.001). E. The mRNA levels of TauT in visceral fat tissues of ob/ob or db/db mice. Mice at the age of 12 weeks and age-matched WT controls were excised and extracted for RNA (n=5, *p<0.05, ***p<0.001). F. The mRNA levels of TauT in subcutaneous fat tissues of high-fat fed mice. Mice at the age of 8 weeks were fed with high fat diet for 2 months. Then mice were excised and extracted for RNA (n=5, ***p<0.05). G. The mRNA levels of TauT in visceral fat tissues of high-fat fed mice. Mice at the age of 8 weeks were fed with high fat diet for 2 months. Then RNA was extracted from the tissues (n=5, ***p<0.001). All Western blots were independently repeated three times, and RT-PCR data are presented as the means ± SD.

TauT promoted the adipogenic differentiation of hASCs. A. The mRNA expression of adipocyte differentiation markers in TauT silencing cells. hASCs transfected with the control or TauT siRNAs and subsequently treated with or without adipogenic inducer for 3 days and cells were harvested for RT-PCR (n=3, *p<0.05, **p<0.01). B. The protein expression of FABP4 in TauT silencing cells in the presence or absence of inducer. Cells treated with or without adipogenic inducer for 3 days and cells were harvested for western blot. The numbers below FABP4 bands were relatively grayscale values qualified by Image J compared to GAPDH. C. The intracellular lipid accumulation in TauT silencing cells. hASCs transfected with the control or TauT siRNAs every 3 days and subsequently induced adipogenic differentiation. Oil Red O staining was performed at day 9. D. The intracellular triglyceride content in TauT silencing cells. E. The mRNA expression of adipocyte differentiation markers in TauT overexpression cells. hASCs were treated with or without adipogenic inducer for 3 days and cells were harvested for RT-PCR (n=3, *p<0.05, **p<0.01). F. The protein expression of FABP4 in TauT overexpression cells in the presence or absence of inducer. hASCs were treated with or without adipogenic inducer for 3 days and cells were harvested for western blot. G. The intracellular lipid accumulation in TauT overexpression cells. hASCs transfected with the control or TauT overexpression plasmid and subsequently induced adipogenic differentiation. Oil Red O staining was performed at day 9. H. The intracellular triglyceride content in TauT overexpression cells. All Western blots were independently repeated three times, and RT-PCR data are presented as the means ± SD.

Hypotaurine and β-alanine promoted the adipogenic differentiation of hASCs, while taurine inhibited. A. The expression of adipocyte differentiation markers in the presence or absence of hypotaurine. hASCs were treated with or without adipogenic inducer and hypotaurine for 3 days and cells were harvested for western blot and RT-PCR (n=3, *p or p<0.05, **p <0.01, ***p<0.001). B. The expression of adipocyte differentiation markers in the presence or absence of β-alanine. hASCs were treated with or without adipogenic inducer and β-alanine for 3 days and cells were harvested for western blot and RT-PCR (n=3, *p or p <0.05, **p or p <0.01, ***p<0.001). C. The expression of adipocyte differentiation markers in the presence or absence of taurine. hASCs were treated with or without adipogenic inducer and taurine for 3 days and cells were harvested for western blot and RT-PCR (n=3, nsp>0.05, *p or p <0.05, **p<0.01, ***p<0.001). D. The intracellular lipid accumulation and triglyceride content in the presence or absence of hypotaurine, taurine or β-alanine. hASCs were treated with hypotaurine(5mM), β-alanine(5mM) or taurine(5mM) and induced adipogenic differentiation. Oil Red O staining was performed at day 9. All Western blots were independently repeated three times, and RT-PCR data are presented as the means ± SD.

The inhibitory effect of TauT knockdown on hASCs adipogenic differentiation could be reversed by hypotaurine and β-alanine, but not taurine. A. The mRNA expression of adipocyte differentiation markers in TauT knockdown cells treated with hypotaurine, taurine or β-alanine. hASCs transfected with the control or TauT siRNAs and subsequently induced adipogenic differentiation. Meanwhile, 5mM hypotaurine, taurine or β-alanine was added. After 3 days, cells were harvested for RT-PCR (n=3, nsp>0.05, *p<0.05, **p<0.01, ***p<0.001). B & C. The intracellular lipid accumulation and triglyceride content in TauT knockdown cells treated with hypotaurine, taurine or β-alanine. hASCs transfected with the control or TauT siRNAs every 3 days and subsequently induced adipogenic differentiation and 5mM hypotaurine, taurine or β-alanine was added. Oil Red O staining was performed at day 9(B). Then the content of triglyceride was quantified (C).

TauT regulated the adipogenic differentiation through preventing the downregulation of Wnt/β-catenin signaling. A. The expression of β-catenin in the presence or absence of inducer. B. The expression of β-catenin in TauT silencing cells in the presence or absence of inducer. hASCs transfected with the control or TauT siRNAs and subsequently treated with or without adipogenic inducer for 3 days. C. The expression of β-catenin under inducer and hypotaurine dose points treatment. hASCs were treated with adipogenic inducer and the indicated dose of hypotaurine for 3 days. D. The expression of β-catenin under inducer and β-alanine dose points treatment. hASCs were treated with adipogenic inducer and the indicated dose of β-alanine for 3 days. E. The expression of β-catenin under inducer and taurine dose points treatment. hASCs were treated with adipogenic inducer and the indicated dose of taurine for 3 days. F. The expression of β-catenin in TauT knockdown cells treated with hypotaurine, taurine or β-alanine. hASCs transfected with the control or TauT siRNAs and subsequently induced adipogenic differentiation. Meanwhile, 5mM hypotaurine, taurine or β-alanine was added for 3 days. FABP4 were detected as marker protein of adipogenic differentiation. G. The expression of PPARγ in hypotaurine or β-alanine treated cells in the presence or absence of GSK3β inhibitor. hASCs were treated with hypotaurine or β-alanine and adipogenic inducer for 3 days, 50nM TWS119 was added in the meantime. H. The expression of PPARγ in TauT overexpression cells in the presence or absence of GSK3β inhibitor. The control and TauT overexpression hASCs were treated with or without adipogenic inducer and GSK3β inhibitor for 3 days. All Western blots were independently repeated three times.

Effect of AS-IV on apoptosis in human umbilical vein endothelial cells stimulated with high glucose conditions. (A) Apoptotic status was determined using a fluorescent microscope (magnification, x200). (B) Apoptotic rates were assessed using annexin V-FITC and PI staining analysis.

Effect of AY 9944-A-7 (30 mmol/L) or ketoconazole (20 mmol/L) on c-mos, MEK, ERK1 and ERK2 mRNA relative expression at 0, 12, 24, 36, and 44 h of IVM in porcine oocytes.(A) Expression of c-mos;

Effect of AY 9944-A-7 (30 mmol/L) or ketoconazole (20 mmol/L) on related protein expression in MAPK pathway at different times of IVM in porcine oocytes. (A) Western blot results of c-mos, MEK1, MEK2 and ERK1/2 protein of AY 9944-A-7 and ketoconazole group at each time. (B) Relative expression levels of c-mos protein in porcine oocytes cultured with AY 9944-A-7 (30 mmol/L) or ketoconazole (20 mmol/L) at different times; (C) Relative expression levels of MEK1 protein; (D) Relative expression levels of MEK2 protein; (E)Relative expression levels of ERK1/2 protein. The values are expressed as mean ± SEM. The data of the same group at different times marked with different letters are significantly different (P < 0.05). The data of different groups at the same time are marked with asterisk to indicate their significant difference (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Te-S-S-NHC-Ru induced apoptosis on MCF-7 (PR+) and MDA-MB-231 (PR-) cell lines.(A) Cancer cells were stained with FITC-AnnexinV and PI, and assessed by a flow cytometer to measure the apoptosis. (B) Quantification of FITC-positive cells. Results were obtained from triplicate separate determinations and the bars represented mean ± S.D. ***P <0.001.

AR treatment improved colitis in Il-10-/- mice (A) The mean DAI values of the AR group were lower than those of the Il-10-/- group during the 2nd week after AR treatment. (B) AR treatment could alleviate the histological manifestations of experimental colitis, as shown by H&E staining. The arrow indicates an inflamed area in the colitis tissue. (C) Histological inflammation scores show that administration of AR clearly decreased inflammation in tissue samples from AR-treated Il-10-/- mice (AR) compared with those from untreated Il-10-/- mice. (D) The protein levels of IL-1β, TNF-α and IL-17A were clearly decreased in colon samples from the AR group compared with those from the untreated Il-10-/- group. (E) The mRNA levels showed the same trend as the protein levels. AR, clematichinenoside AR; WT, wild-type; IL-17A, interleukin 17A; TNF-α, tumor necrosis factor-α; and IL-1β, interleukin 1β; DAI, disease activity index. The experiments were performed at least 3 times independently with 6 to 8 mice in each group, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05.

AR treatment improved the expression of junctional molecules (A-B) IF showed that the expression of the TJ proteins occludin and ZO-1 (green) was inhibited in the intestinal mucosal epithelium. DAPI was used to stain nuclei (blue), and the merged image shows the overlay of TJ protein and DAPI staining. (C-D) The protein levels of ZO-1 and occludin in the intestinal mucosa of the AR group were clearly lower than those in the intestinal mucosa of the untreated Il-10-/- group and was similar to those in the WT group. AR, clematichinenoside AR; WT, wild-type; TJ, tight junction; IF, immunofluorescence; DAPI, 4′,6-diamidino-2-phenylindole; and NS, no significance. The experiments were performed at least 3 times independently with 6 mice in each group, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05.

AR treatment reduced the permeability of the intestines of Il-10-/- mice (A) The intestinal permeability detection results indicated that the serum dextran conjugate levels of the AR group were lower than those of the Il-10-/- group and similar to those of the WT group. (B-C) AR-treated mice had lower rates of bacterial translocation in the MLN and liver than did the untreated Il-10-/- mice and had similar rates as the WT mice. AR, clematichinenoside AR; WT, wild-type; and MLN, mesenteric lymph node. The experiments were performed at least 3 times independently with 6 mice in each group, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05.

AR treatment reduced the apoptosis rate of epithelial cells in Il-10-/- mice(A-B) TUNEL staining showed that the AR group had fewer apoptotic cells in the intestinal epithelium than did the Il-10-/- group and a similar number as the WT group. The arrow indicates TUNEL-positive cells. (C-D) The expression of the antiapoptotic factor Bcl-2 was higher in the AR group than in the Il-10-/- group but was lower than that in the WT group. However, the expression levels of Bax and cleaved caspase-3 were lower in the AR group than in the Il-10-/- group, and AR-treated Il-10-/- mice showed significant decreases in Bax and cleaved caspase-3 expression; however, the expression levels were still higher than those in the WT group. AR, clematichinenoside AR; WT, wild-type; and NS, no significance. The experiments were performed at least 3 times independently with 6 mice in each group, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05.

AR treatment reduced Th17 responses and enhanced Treg responses in Il-10-/- mice (A-B) A significant decrease in the percentage of IL-17A+ T cells within the CD4+ T cell population in the spleen was observed in the AR group compared with the Il-10-/- group, but this T lymphocyte proportion was still higher than the proportion in the WT group. (C-D) A significant decrease in the percentage of IL-17A+ T cells within the CD4+ T cell population in the MLN was observed in the AR group compared with the Il-10-/- group, but this T lymphocyte proportion was still higher than the proportion in the WT group. (E-F) A significant increase in the percentage of Foxp3+ T cells (Tregs) within the CD4+ CD25+ T cell population in the spleen was observed in the AR group compared with the Il-10-/- group, but this T lymphocyte proportion was still lower than the proportion in the WT group. (G-H) A significant increase in the percentage of Foxp3+ T cells within the CD4+ CD25+ T cell population in the MLN was observed in the AR group compared with the Il-10-/- group, but this T lymphocyte proportion was still lower than the proportion in the WT group. AR, clematichinenoside AR; WT, wild-type; and MLN, mesenteric lymph node. The experiments were performed at least 3 times independently with 6 mice in each group, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05.

AR treatment partly ameliorated the inflammatory response in Il-10-/- mice by attenuating PI3K/Akt signaling (A-B) Western blot analysis showed that the levels of both p-PI3K and p-Akt were attenuated in the AR group compared with the Il-10-/- group but were still higher than those in the WT group. (C) The mRNA levels showed the same trend as the protein levels. AR, clematichinenoside AR; and WT, wild-type. The experiments were performed at least 3 times independently with 6 mice in each group, and one representative result is shown. The results are presented as the means ± SD. *P < 0.05.

Effects of zeaxanthin dipalmitate (ZD) on hepatic oxidative stress in each group. Hepatic protein expressions of (A) catalase (CAT )

Effects of zeaxanthin dipalmitate (ZD) on hepatic oxidative stress in each group. Hepatic protein expressions of (B) superoxide dismutase 1 (SOD1)