Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 142 Nd cCasp3 D3E9 Fluidigm 3142004A 143 Nd CD19 HIB19 Biolegend 302202 144 Nd pPLCg2 [Y759] K86-689.37 Fluidigm 3144015A 145 Nd CD4 RPA-T4 Fluidigm 3145001B 146 Nd IgD IA6-2 Fluidigm 3146005B 147 Sm CD20 2H7 Fluidigm 3147001B 148 Nd IgA Polyclonal Fluidigm 3148007B 149 Sm CD25 2A3 Fluidigm 3149010B 150 Nd pStat5 [Y694] 47 Fluidigm 3150005A 151 Eu CD123 6H6 Fluidigm 3151001B 153 Eu pStat1 [Y701] 4a Fluidigm 3153005A 154 Sm CD45 HI30 Fluidigm 3154001B 155Gd CD27 L128 Fluidigm 3155001B 156 Gd p38 [T180/Y182] D3F9 Fluidigm 3156002A 157 Gd CD24 ML-5 Biolegend 311102 158 Gd pStat3 [Y705] 4/P-Stat3 Fluidigm 3158005A 159Tb CD11c Bu15 Fluidigm 3159001B 160Gd CD14 M5E2 Fluidigm 3160001B 161 Dy CD141(BDCA-3) AD5-14H12 Miltenyi 130-090-694 162 Dy CD66b 80H3 Fluidigm 3162023B 163 Dy CD56 NCAM16.2 Fluidigm 3163007B 164 Dy IkBa L35A5 Fluidigm 3164004A 165 Ho pCREB [S133] 87G3 Fluidigm 3165009A 166 Er CD16 B73.1 Biolegend 360702 167 Er CD38 HIT2 Fluidigm 3167001B 168 Er CD8 SK1 Fluidigm 3168002B 169 Tm CD45RA HI100 Fluidigm 3169008B 170 Er CD3 UCHT1 Fluidigm 3170001B 171 Yb pERK1/2 [T202/Y204] D13.14.4E Fluidigm 3171010A 172 Yb Anti-Ki-67 B56 Fluidigm 3172024B 174 Yb HLA-DR L243 Fluidigm 3174001B 175Lu CD7 CD7-6B7 Biolegend 343102 176 Yb CD127/IL-7Ra P48-48 Novus Bio MAB306-100 209Bi CD11b/Mac-1 ICRF44 Fluidigm 3209003B Open in a separate window 1 Open channels are not shown but include Pd channels, Cd channel, Pt channels, 89Y,152Sm and 173Yb Whole blood phosphoflow panel1.

Techniques:

Journal: Cell Chemical Biology

Article Title: Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

doi: 10.1016/j.chembiol.2021.05.002

Figure Lengend Snippet: The effect of N-terminal NanoLuciferase, HaloTag, or SNAP-tag additions to IL-23 receptor subunits on IL-23-induced STAT3 phosphorylation STAT3 phosphorylation induced in HEK293T cells transfected with different tagged variants of the IL-23 receptor after a 30-min incubation with increasing concentrations of IL-23. Data are expressed as a percentage of the response obtained with 15 nM IL-23. Values are mean ± SEM from four or three (NL-IL23R and HT-IL12Rβ1) independent experiments.

Article Snippet: AlphaLISA SureFire Ultra p-STAT3 (Tyr705) Assay Kit , Perkin Elmer , Cat# ALSU-PST3.

Techniques: Transfection, Incubation

Journal: Cell Chemical Biology

Article Title: Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

doi: 10.1016/j.chembiol.2021.05.002

Figure Lengend Snippet:

Article Snippet: AlphaLISA SureFire Ultra p-STAT3 (Tyr705) Assay Kit , Perkin Elmer , Cat# ALSU-PST3.

Techniques: Recombinant, Modification, Staining, Purification, Luciferase, Expressing, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Acetylcholine Acts on Androgen Receptor to Promote the Migration and Invasion but Inhibit the Apoptosis of Human Hepatocarcinoma

doi: 10.1371/journal.pone.0061678

Figure Lengend Snippet: (A–D) Western blots showed the effects of Ach and MEC on STAT3 phosphorylation (A, B) and Akt phosphorylation (C, D) and in SNU-449 cells. Phosphorylation of STAT3 (A) and Akt (C) was enhanced by Ach in SNU-449 cells, whereas acetylcholine receptor antagonists MEC blocked the effect of Ach on STAT3 (B) and AKT (D) phosphorylation.

Article Snippet: Primary antibodies used here included rabbit monoclonal anti-AR (D6F11) (3H8; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-nicotinic acetylcholine receptor alpha 7 (ab10096, abcam, USA), rabbit monoclonal anti-pSTAT3, anti-pAKT (195-14; BioCheck, Foster City, CA), and monoclonal anti-β-actin (Sigma).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. Forty-eight hours after the transfection, OA chondrocytes were serum starved and then stimulated with or without IL-1β (10 ng/ml) for 48 h. (A). At end of treatment, cell lysates were prepared using RIPA buffer and protein levels of cleaved caspase-3, cleaved caspase-8, pro-caspase-9, cleaved PARP and Bid were investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. (B) Fold change relative to β-actin for the expression these proteins were quantified by measuring specific signal intensities using ImageJ software. *p≤0.05, as compared to pcDNA, # ≤0.05, as compared to pcDNA+IL-1β. (C) After transfection, OA chondrocyte were stimulated IL-1β (1 ng/ml) for 30 minutes at 37°C and then stained with JC-1 (2.5 μM) and mitochondrial membrane potential loss was measured by ratio of red to green fluorescence. Values were expressed relative to unstimulated control and graph shows mean ± SD of four replicates from a representative experiment and three such independent experiments were carried out. *p≤0.05, as compared to control, # ≤0.05, as compared to IL-1β. (D) Cytosolic lysates were prepared after 48 h of IL-1β (10 ng/ml) stimulation in transfected chondrocytes and levels of cytosolic cytochrome-c was estimated by immunoblotting using validated antibody. β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals.

Article Snippet: Luciferase reporter assay Chondrocytes were transfected with pNrf2/ARE-Luc reporter vector (LR-2016, Signosis, Santa Clara, CA, USA) and pRL-TK (10 nM) as internal control by Nucleofection as described above.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Software, Staining, Fluorescence

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Primary human chondrocytes were isolated from cartilage of OA patients. (A) Nrf2/ARE luciferase reporter vector was transfected in OA chondrocytes and 48 hours later, chondrocytes were stimulated with IL-1β (1 ng/ml) for indicated time and luciferase activity was measured by Dual-Glo® reporter assay. Renilla luciferase was cotransfected for normalization purposes. Values are the mean±SD of 2 experiments each performed in triplicate. (B–C) Human OA chondrocytes were stimulated with IL-1β (1 ng/ml) for indicated time. At the end of treatment, chondrocytes were harvested and cell lysates were prepared using RIPA buffer for immunoblot analysis. (B) Protein expression of Nrf2, HO-1, NQO1 and SOD2 was investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. (C) Fold change relative to β-actin for the expression these proteins were quantified by ImageJ software. *p≤0.05, as compared to control. (D) Human OA chondrocytes were pre-treated with anti-oxidants-NAC(10 mM) or GSH (10 mM) or DPI (5μM) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h and RNA were isolated from harvested chondrocytes and expression of Nrf2 and HO-1 was measured by quantitative PCR using the SYBR® green assay (Life Technologies). β-actin was used as endogenous expression control. Bar graph represents mean±SD from three subjects. *p≤0.05, as compared to control, # ≤0.05, as compared to IL-1β.

Article Snippet: Luciferase reporter assay Chondrocytes were transfected with pNrf2/ARE-Luc reporter vector (LR-2016, Signosis, Santa Clara, CA, USA) and pRL-TK (10 nM) as internal control by Nucleofection as described above.

Techniques: Isolation, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Reporter Assay, Western Blot, Expressing, Software, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with siRNA specific for Nrf2 (siNrf2, 100 nM) or negative controls siRNA (siCNT, 100 nM) using X-tremeGENE™ siRNA transfection reagent or transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. (A, B) Forty-eight hours after the transfection with (A) siRNA or (B) expression vector, OA chondrocytes were stained with DHR123 (5 μM) for 0.5 h at 37°C, and stimulated with IL-1β (1 ng/ml) for 30 minutes at 37°C and ROS generation was estimated by measuring fluorescence emission at 535 nm. Bar graph shows arbitrary fluorescence units indicating ROS levels. (C–D) Forty-eight hours after the transfection with (C) siRNA or (D) expression vector, OA chondrocytes were stained with stimulated with IL-1β (1 ng/ml) for 30 minutes at 37°C, supernatants were collected and generation of H2O2 in the supernatant was estimated using Amplex red assay as described in method section. Data points represent mean±SD of three replicates from a representative experiment and three such independent experiments were carried out.*p≤0.05, as compared to control, # ≤0.05, as compared to IL-1β.

Article Snippet: Luciferase reporter assay Chondrocytes were transfected with pNrf2/ARE-Luc reporter vector (LR-2016, Signosis, Santa Clara, CA, USA) and pRL-TK (10 nM) as internal control by Nucleofection as described above.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Fluorescence, Amplex Red Assay

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. Forty-eight hours after the transfection, OA chondrocytes were serum starved and then stimulated with or without IL-1β (10 ng/ml) for 48 h. (A) Cell viability was measured by MTT assays using method described in method sections. Unstimulated chondrocytes were served as control and viability was expressed relative to control cells. Data point was expressed as a mean ± SD of two replicates from a representative experiment and three such independent experiments were carried out. *p≤0.05, as compared to pcDNA (control), # ≤0.05, as compared to pcDNA+IL-1β group. (B) Apoptosis was estimated by TUNEL assay as described in method section. Flow cytometric histograms showing percent TUNEL positive chondrocytes were shown. Twenty thousand cells in each group were acquired using a flowcytometer and percent apoptosis was calculated using flow Jo software and expressed as a mean±SD of three replicates from a representative experiment and three such independent experiments were carried out. (C) Apoptosis was visualized by DNA fragmentation assay as described in method section and DNA samples were electrophoresed in 1.8% agarose gel and DNA fragmentation pattern in control and simulated groups were visualized by EtBr staining. Results are representatives of three experiments performed on samples obtained from three individuals.

Article Snippet: Luciferase reporter assay Chondrocytes were transfected with pNrf2/ARE-Luc reporter vector (LR-2016, Signosis, Santa Clara, CA, USA) and pRL-TK (10 nM) as internal control by Nucleofection as described above.

Techniques: Transfection, Expressing, Plasmid Preparation, TUNEL Assay, Software, DNA Fragmentation Assay, Agarose Gel Electrophoresis, Staining

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. Forty-eight hours after the transfection, OA chondrocytes were serum starved and then stimulated with or without IL-1β (10 ng/ml) for 15 and 30 minutes at 37°C (A–C). At end of treatment, cell lysates were prepared using RIPA buffer and immunoblotting was performed to detect the protein levels of (A) p-ERK1/2 (Thr180/Tyr182), total ERK1/2, p-MEK1/2 (Ser217/221), total MEK1/2, p-ELK-1 (Ser383), p-P70S6K (Ser411), p-P90RSK (Ser380). β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. (B–C) At end of transfection, chondrocytes were treated with ERK inhibitor PD98059 (30 μM) for 2 h and then stimulated with IL-1β (10 ng/ml) for (B) 15 minutes and (C) 16 h at 37°C and processed for (B) immunoblotting with p-ERK1/2 and total ERK1/2 and (C) TUNEL assay as described in method section. Bar graph shows mean±SD of three replicates from a representative experiment and three such independent experiments were carried out. *p≤0.05, as compared to pcDNA, # ≤0.05, as compared to pcDNA+IL-1β, $≤0.05, as compared to pNrf2+IL-1β.

Article Snippet: Luciferase reporter assay Chondrocytes were transfected with pNrf2/ARE-Luc reporter vector (LR-2016, Signosis, Santa Clara, CA, USA) and pRL-TK (10 nM) as internal control by Nucleofection as described above.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, TUNEL Assay

Journal: Stem Cell Research & Therapy

Article Title: The adipose-derived mesenchymal stem cell secretome promotes hepatic regeneration in miniature pigs after liver ischaemia-reperfusion combined with partial resection

doi: 10.1186/s13287-021-02284-y

Figure Lengend Snippet: Effects of ASCs and the ASC-secretome on liver regeneration-related proteins. a Immunohistochemical staining of the Ki67 protein in liver tissue sections at all time points in the four groups. b Results of Ki67 immunohistochemical analysis. c Western blot of liver regeneration-related proteins. d Analysis of PCNA protein expression levels. Figure 4e: Analysis of p-STAT3 protein expression levels. f Analysis of SOCS3 protein expression levels. The data are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, versus the saline group. # p < 0.05, ## p < 0.01, versus the DMEM group.

Article Snippet: The membrane was blocked in TBST containing 5% nonfat powdered milk for 2 h at room temperature to prevent nonspecific binding, incubated with primary antibody overnight at 4 °C, and then incubated with a horseradish peroxidase (HRP)-conjugated anti-species secondary antibody for 2 h. Antibodies against PCNA (Abcam, Cambridge, UK), p-STAT3, STAT3, suppressor of cytokine signalling 3 (SOCS3) (ImmunoWay, Plano, USA) and β-actin and a horseradish peroxidase (HRP)-conjugated antibody (Sangon Biotech, Shanghai, China) were used.

Techniques: Immunohistochemical staining, Staining, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: The adipose-derived mesenchymal stem cell secretome promotes hepatic regeneration in miniature pigs after liver ischaemia-reperfusion combined with partial resection

doi: 10.1186/s13287-021-02284-y

Figure Lengend Snippet: The experimental procedure and the mechanism by which the ASC-secretome promotes liver regeneration. The model of liver ischaemia-reperfusion combined with partial hepatectomy was established by laparoscopy. The ASC-secretome of miniature pigs was prepared and injected into the liver parenchyma immediately after the operation. The ASC-secretome exerted obvious anti-inflammatory and liver regenerative effects. The mechanism may be as shown in Fig. 5. After partial hepatectomy, the STAT3 signalling pathway was activated to promote the proliferation of hepatocytes. Furthermore, a SOCS3 negative feedback loop was activated to terminate the STAT3 signalling cascade. The ASC-secretome acted on SOCS3 to reduce its expression and interrupted the negative feedback regulation of the STAT3 signalling pathway, accelerating the process of liver regeneration. Cytokines in the ASC-secretome regulate inflammation and promote liver regeneration.

Article Snippet: The membrane was blocked in TBST containing 5% nonfat powdered milk for 2 h at room temperature to prevent nonspecific binding, incubated with primary antibody overnight at 4 °C, and then incubated with a horseradish peroxidase (HRP)-conjugated anti-species secondary antibody for 2 h. Antibodies against PCNA (Abcam, Cambridge, UK), p-STAT3, STAT3, suppressor of cytokine signalling 3 (SOCS3) (ImmunoWay, Plano, USA) and β-actin and a horseradish peroxidase (HRP)-conjugated antibody (Sangon Biotech, Shanghai, China) were used.

Techniques: Injection, Expressing