Journal: Frontiers in Immunology

Article Title: Identification and verification of the optimal feature genes of ferroptosis in thyroid-associated orbitopathy

doi: 10.3389/fimmu.2024.1422497

Figure Lengend Snippet: Primer name and sequence.

Article Snippet: The PVDF membrane was then incubated overnight on a shaking incubator at 4°C in ACO1 antibody (PA5-41753, Thermofisher), HCAR1 antibody (ER62150, HuaBio) and MMD antibody (A06022, Boster) prepared according to the corresponding dilution ratio.

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Identification and verification of the optimal feature genes of ferroptosis in thyroid-associated orbitopathy

doi: 10.3389/fimmu.2024.1422497

Figure Lengend Snippet: Differential expression and ROC curve of OFGs of ferroptosis. (A) The expression difference of ACO1 between TAO and Normal. (B) The expression difference of HCRA1 between TAO and Normal. (C) The expression difference of MMD between TAO and Normal. (D) The predictive value of ACO1 in TAO from the ROC curve. (E) The predictive value of HCRA1 in TAO from the ROC curve. (F) The predictive value of MMD in TAO from the ROC curve. Each panel displayed the AUC under the curve and 95% CI. ROC, ROC curve; AUC, area under the curve; CI, confidence interval.

Article Snippet: The PVDF membrane was then incubated overnight on a shaking incubator at 4°C in ACO1 antibody (PA5-41753, Thermofisher), HCAR1 antibody (ER62150, HuaBio) and MMD antibody (A06022, Boster) prepared according to the corresponding dilution ratio.

Techniques: Quantitative Proteomics, Expressing

Journal: Frontiers in Immunology

Article Title: Identification and verification of the optimal feature genes of ferroptosis in thyroid-associated orbitopathy

doi: 10.3389/fimmu.2024.1422497

Figure Lengend Snippet: GSEA analysis of OFGs for ferroptosis in TAO. (A) GSEA analysis of ACO1. (B) GSEA analysis of HCRA1. (C) GSEA analysis of MMD.

Article Snippet: The PVDF membrane was then incubated overnight on a shaking incubator at 4°C in ACO1 antibody (PA5-41753, Thermofisher), HCAR1 antibody (ER62150, HuaBio) and MMD antibody (A06022, Boster) prepared according to the corresponding dilution ratio.

Techniques:

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Positive allosteric effects of protopanaxadiol ginsenosides on P2X7. (A) A YO-PRO-1 iodide uptake assay was used to determine the effect of multiple ginsenosides on human P2X7 stably expressed in HEK-293 cells. Agonist (ATP, 200 μM) and modulator (10 µM) or vehicle (veh) were premixed at 10× final concentration and auto-injected together (coinjection) using a Flexstation 3 multimode plate reader. YO-PRO-1 dye uptake (relative fluorescence units (RFU)) was measured over 300 seconds. Data are expressed as area under curve means ± S.D. (B) Dose-response curves to ATP in the absence and presence of 10 µM ginsenoside-CK, -Rd, or -Rb1 or the aglycone PPD with a four-parameter nonlinear regression. Data are collated from three independent experiments each performed in triplicate. Error bars represent S.D. (C) Chemical structures for each of the ginsenoside positive modulators and the inactive aglycone PPD are shown.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Stable Transfection, Concentration Assay, Injection, Fluorescence

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Structural details of tested glycosides Substitution pattern is derived from Fig. 1C . Maximum response was defined at 1 mM ATP. To calculate the EC 50 ratio, the average EC 50 value for ATP + vehicle was used from each set of experiments. Average EC 50 value is shown with 95% confidence intervals in parentheses.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Derivative Assay

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Screening glycosides containing disaccharide and trisaccharide moieties at P2X7. (A) Initial experiments used a fixed concentration of ATP (200 µM final) and glycoside (10 µM final) to screen selected glycosides at P2X7. Data are collated from two to four independent experiments. Ginsenoside-CK was used as the control PAM and is shown in blue. YO-PRO-1 uptake was measured as area under curve (50–300 seconds), and data are expressed as percentage of control, where the control is ATP + DMSO. One-way ANOVA with Dunnett’s multiple comparisons test was performed. * P < 0.05 compared with DMSO control. (B) Dose-response curve to ATP in the presence of vehicle (DMSO), ginsenoside-CK, or gypenoside XVII (10 µM). (C) Dose-response curve to ATP in the presence of vehicle (DMSO), ginsenoside-CK, or gypenoside XLIX (10 µM). (D) Dose-response curve to ATP in the presence of vehicle (DMSO), ginsenoside-CK, or saikosaponin A (10 µM). Data points are means ± S.D. The same curves from DMSO and ginsenoside-CK are shown in (B and D). (E) Summary of data from YO-PRO-1 uptake experiments in HEK-hP2X7 cells or parental nontransfected HEK-293 cells in response to drug alone (saikosaponin A or solanine). (F) Lack of effect of the P2X7-selective antagonist AZ10606120 (AZ106; 10 µM) on ATP-induced YO-PRO-1 uptake when solanine or saikosaponin were used. One-way ANOVA with Sidak’s multiple comparisons test was performed. * P < 0.05, ns denotes not significant.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Concentration Assay

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Screening glycosides containing monosaccharide moieties at P2X7. Dose-response curves to ATP in the presence of vehicle (DMSO), ginsenoside-CK, and the following: daucosterol (10 µM) (A), stevioside (SV; 10 µM) (B), ginsenoside-F1 (F1; 10 µM) (C), ginsenoside-F2 (F2; 10 µM) (D), ouabain (10 µM) (E), or scilliroside (10 µM) (F). Ginsenoside-CK is demonstrated throughout as the reference compound (same data shown in each plot). Data are collated from three independent experiments each performed in triplicate. Error bars represent S.D.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques:

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Molecular docking of ginsenoside-CK and ginsenoside-F1 to the central vestibule pocket of hP2X7. Ginsenoside-CK (cyan) docked into the central vestibule in the ATP-bound homology model of human P2X7 and right, ginsenoside-F1 (green) docked into the same site. Side chains of amino acid residues of hP2X7 implicated in interactions are shown.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques:

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Diastereoisomers of ginsenosides have different activity at hP2X7. (A) Dose-response curves to ATP in the presence of vehicle (DMSO), 20( S )-ginsenoside-Rg3, or 20( R )-ginsenoside-Rg3 (10 µM). (B) Dose-response curves to ATP in the presence of vehicle (DMSO), ginsenoside-20( S )-Rh2, or ginsenoside-20( R )-Rh2 (10 µM). Data are collated from three independent experiments and are means ± S.D. The same DMSO data are shown in both plots. (C) Chemical structures of ginsenoside-Rg3 and ginsenoside-Rh2 are shown with the stereo-centers highlighted in red.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Activity Assay

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Induced-fit docking of 20S-Rg3 at human P2X7 ginsenoside-20( S )-Rg3 (green) docked into the central vestibule site in a homology model of ATP-bound human P2X7 (open state). The predicted orientation of the stereocentre C-20 is such that the –OH is pointing away from the hydrophobic face of the binding site, thus minimizing any repulsive interactions. Key side chains of residues D318, L320, and F322 are indicated.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Binding Assay

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: The PAM effects of glycosides in human THP-1 monocyte cell line. (A) ATP-induced calcium responses were measured in fura-2 AM loaded THP-1 cells in suspension using a Flexstation 3 plate reader. Agonist (500 µM ATP) and PAM (ginsenoside-CK 10 µM) were coinjected after establishment of a baseline for 40 seconds. Cells were preincubated with various P2X7 antagonists for 10 minutes prior to start of plate recordings. (B) Summary of collated data from calcium measurements. Fura-2 responses were calculated as area under curve. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test to assess the effect of antagonists. * P < 0.05. (C) Investigating the P2X7 dependence of glycoside effects on ATP-induced calcium responses. AZ10606120 (AZ106; 10 µM) was added to cells to block P2X7 receptors prior to measuring calcium responses. Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test comparing each column against the control (500 µM ATP + DMSO). * P < 0.05. (D) Cell viability experiments were performed over 24 hours using HEK-hP2X7 cell line. Alamar blue fluorescence was measured, and data were normalized to percentage of control (DMSO). Data were collated from five independent experiments. One-way ANOVA was used to analyze the data with Sidak’s multiple comparisons test to compare selected pairs of columns (DMSO + ATP vs. ginsenoside + ATP). * P < 0.05. (E) Cell viability experiments were performed over 24 hours using THP-1 cells. Alamar blue fluorescence was measured and data were normalized to percentage of control (DMSO). Data were collated from five independent experiments. One-way ANOVA was used to analyze the data with Sidak’s multiple comparisons test to compare selected pairs of columns (DMSO + ATP vs. ginsenoside + ATP). * P < 0.05.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Blocking Assay, Fluorescence

Journal: Molecular Pharmacology

Article Title: Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

doi: 10.1124/molpharm.120.000129

Figure Lengend Snippet: Proposed structure-activity relationship for glycosides acting as PAMs at P2X7. (A) The chemical structure of ginsenoside-CK is shown with important groups highlighted. Glucose attachment (cyan) is critical for activity at P2X7. C-6 substitutions are not tolerated (yellow). (B) The chemical structure of ginsenoside-20( S )-Rg3 is demonstrated with positioning relative to the P2X7 binding mode (inverted). The C-3 glucose attachments (cyan) face up into the binding pocket. The C-20 hydroxyl group shows that stereochemistry and positioning are critical for activity at P2X7.

Article Snippet: Ginsenoside-CK (CAS 39262-14-1), ginsenoside-Rb1 (CAS 41753-43-9), ginsenoside-Rd (CAS 52705-93-8), 20( S )-ginsenoside-Rg3 (CAS 14197-60-5), 20( R )-ginsenoside-Rh2 (CAS 112246-15-8), protopanaxadiol (PPD) (CAS 30636-90-9), glycyrrhizic acid (CAS 1405-86-3), stevioside (CAS 57817-89-7), daucosterol (CAS 474-58-8), esculentoside A (CAS 65497-07-6), mogroside V (CAS 88901-36-4), saikosaponin A (CAS 20736-09-8), and stevenleaf (CAS 80321-63-7) were from Shanghai Richem International Ltd., China (supplier code CDCMANSETE).

Techniques: Activity Assay, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: G?i2 Signaling Is Required for Skeletal Muscle Growth, Regeneration, and Satellite Cell Proliferation and Differentiation

doi: 10.1128/MCB.00957-13

Figure Lengend Snippet: Knockdown of Gαi2 results in decreased proliferation, fusion, and differentiation. (A) Satellite cells were isolated and transfected with siGαi2 or siCtrl, cultured in growth medium for 3 days, and then pulsed with 10 μM BrdU for 8 h. Cell proliferation was determined by immunostaining using antibodies against BrdU (green); DAPI-counterstained nuclei are in blue. The histogram represents the percentage of BrdU incorporation, and the data are shown as the means and SEM. from three independent experiments. ***, P < 0.001. (B) Satellite cells were isolated, cultured in differentiation medium for 48 h, and then infected with an shRNA directed against Gαi2 (shGαi2) or shRNA control (shGFP). The cells were cultured for an additional 72 h, fixed, and stained with an antibody to MyHC (green) and DAPI (blue). The diameter and the fusion index (number of nuclei per myotube) of MyHC-stained myotubes were determined. The results are the means and SEM. ***, P < 0.001; ****, P < 0.0001.

Article Snippet: A small interfering RNA (siRNA) negative-control construct (On-Targetplus nontargeting control siRNA [D-001810]) and siGαi2 (sc-41753) were purchased from Thermo Scientific.

Techniques: Knockdown, Isolation, Transfection, Cell Culture, Immunostaining, BrdU Incorporation Assay, Infection, shRNA, Control, Staining

Journal: Molecular and Cellular Biology

Article Title: G?i2 Signaling Is Required for Skeletal Muscle Growth, Regeneration, and Satellite Cell Proliferation and Differentiation

doi: 10.1128/MCB.00957-13

Figure Lengend Snippet: Gαi2 regulates the expression of muscle-specific miRNAs in satellite cells and in C2C12 myoblasts via PKC. (A) Satellite cells transfected with siGαi2 or siCtrl or infected with AdEV or AdGαi2(Q205L) were cultured in differentiation medium for 72 h. Expression of muscle-specific miRNAs was analyzed by qRT-PCR. *, P < 0.05; **, P < 0.01. (B) C2C12 myoblasts were pretreated or not for 1 h with 1 μM PKCi, infected with AdEV or AdGαi2(Q205L), and cultured in differentiation medium for an additional 48 h. Expression of miR-1 was analyzed by qRT-PCR. n = 3; ***, P < 0.001 versus AdEV; ##, P < 0.01 versus vehicle control-treated cells. The error bars indicate SEM.

Article Snippet: A small interfering RNA (siRNA) negative-control construct (On-Targetplus nontargeting control siRNA [D-001810]) and siGαi2 (sc-41753) were purchased from Thermo Scientific.

Techniques: Expressing, Transfection, Infection, Cell Culture, Quantitative RT-PCR, Control