Journal: STAR Protocols

Article Title: Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models

doi: 10.1016/j.xpro.2024.103274

Figure Lengend Snippet:

Article Snippet: CD326 (EpCAM) antibody, anti-human, PE, REAfinity (flow cytometry 1:200) , Miltenyi Biotec , Cat# 130-110-999; RRID: AB_2657495.

Techniques: Immunofluorescence, Flow Cytometry, Control, Recombinant, Red Blood Cell Lysis, Saline, Software, Cell Culture, Plasmid Preparation, Microscopy

Journal: Frontiers in Immunology

Article Title: Anti-inflammatory mechanism of total flavonoids from Polygala fallax Hemsl. based on network pharmacology, molecular docking, and experimental validation

doi: 10.3389/fimmu.2025.1690388

Figure Lengend Snippet: (A) Expression of INS protein in RAW264.7 cells induced by PFHF (×400, scale: 25μm); (B) Expression of CASP3 protein in RAW264.7 cells induced by PFHF (×400, scale: 25μm); (C) Expression of Cox-2 protein in RAW264.7 cells induced by PFHF (×400, scale: 25μm); (D) iNOS protein expression in RAW264.7 cells induced by PFHF (×400, scale: 100μm); (E) Expression of INS and CASP3 protein in RAW264.7 cells induced by PFHF (n=3). (F) Expression of Cox-2 and iNOS protein in RAW264.7 cells induced by PFHF (n=3). ns, Not significant. **P<0.01, ****P< 0.0001.

Article Snippet: PFH tablet (purchased from Pharmacy of Guangxi International Zhuang Medical Hospital, batch No.: 20230901); Mouse macrophage RAW264.7 (purchased from Zhongsheng Beikong Biotechnology Co., LTD., identified by STR); INS antibody (Proteintech, 001048883), CASP3 antibody (Proteintech, 00136098), i-NOS antibody (Abcam, GR3240243-2), Cox-2 antibody (Abcam, 00136098) GR3381592-5), donkey anti-rabbit fluorescent secondary antibody (Abcam, GR3360238-2); CCK-8 kit (meilunbio, MA0218-3-Mar-05G); IL-6 mouse ELISA test kit (shfksc, F2163-A), TNF-α mouse ELISA test kit (shfksc, F2132-A), IL-1β mouse ELISA test kit (shfksc, F2040-B).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Relationship between NKG2C + ANK cells and COVID-19: Scatter dot with bar plot showing expression of NKG2C + ANK cells at baseline in the overall cohort (no infection, n=64 and infection, n=16), control group (no infection, n=20 and infection, n=10) and Mw group (no infection, n=44 and infection, n=6) with respect to SARS-CoV-2 infection. Black and red upside shaded triangles represent NKG2C + ANK for Control group and Mw group respectively and unshaded upside triangles represent NKG2C + ANK of both groups combined. ***p < 0.001, **p < 0.01 and *P < 0.05.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Infection, Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Impact of Mw on both ANK cells and iNK) cells: (A) Scatter dot with bar plot showing the expression of NKG2C + ANK cells (Mw group -n=30 and control group- n=15) and (B) NKG2A + iNK cells (Mw group-n=30 and control group- n=15) with and without of Mw vaccine at baseline and day 60. (C) Scatter dot with bar plot showing kinetics of NKG2C + ANK cells, and (D) NKG2A + iNK cells expression in Mw group (n=30) at baseline, day 30, day 60, and day 100. (E) log2FC expression of NKG2C + and NKG2A + in both Mw group (n=30) and vontrol group (n=15) at day 60 after normalization with baseline. Red upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for Mw group. Black upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for control group. ****p < 0.0001, **p <0.01, ***p <0.001, and ns, p-value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Impact of Mw on upregulation of NKG2C + ANK cells with respect to NKG2C expression at baseline: Scatter dot with bar plot showing the kinetics of (A) NKG2C + ANK cells (n=15) and (C) NKG2A + iNK (n=15) cells expression in Mw group at baseline, days 30 and 60 in respect to <15% NKG2C at baseline. (B) NKG2C + ANK (n=15) cells and (D) NKG2A + iNK (n=15) cell expression in Mw group at baseline, days 30 and 60 in respect to >15% NKG2C at baseline. Log2FC expression of (E) NKG2C + ANK and (F) NKG2A + iNK cells at day 60 after normalization with baseline value in Mw group (>/<15%, n=15) and control group. Red upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK respectively for Mw group. Black upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for control group. ****p < 0.0001, **p < 0.01, *p < 0.05, and ns, p-value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Mw showed sustained effect on NKG2C/NKG2A ratios until 100 days and Impact of Mw intracellular cytokine (IFN-γ) release: Scatter dot with bar plot showing (A) NKG2C/NKG2A ratio of control group (n=15) and Mw group (n=30) at baseline and day 60. (B) Kinetics of NKG2C/NKG2A ratio at baseline, days 30, 60, and 100 in Mw group. (C, D) Kinetics of NKG2C/NKG2A ratio at baseline and day 60 on the basis of >/< 15% NKG2C at baseline in Mw group and control group, respectively. (E) intracellular IFN-γ release in both control (n=5) and Mw (n=5) groups at baseline and day 60. Red and black shaded circles represent Mw group and control group, respectively. ****p < 0.0001, *p < 0.05 and ns, p-value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Impact of an Immune Modulator Mycobacterium-w on Adaptive Natural Killer Cells and Protection Against COVID-19

doi: 10.3389/fimmu.2022.887230

Figure Lengend Snippet: Impact of the second dose of Mw on ANK and INK cells: Scatter dot with bar plot showing expression of (A) NKG2C + ANK (day 60, single dose; n=17, double dose; n=12 and day 100, single dose; n=17, double dose; n=12), (B) NKG2A + iNK cells (day 60, single dose; n=12, double dose; n=12 and day 100, single dose; n=17, double dose; n=12) and (C) NKG2C/NKG2A (day 60, single dose; n=17, double dose; n=12 and day 100, single dose; n=17, double dose; n=12) ratio with respect to single and double dose of Mw vaccine at day 60 and 100. Red upside and downside shaded triangles represent NKG2C + ANK and NKG2A + iNK, respectively, for Mw group. Red-shaded circle represents Mw group. ***p < 0.001, *p < 0.05 and ns, p value not significant.

Article Snippet: For surface staining, 0.5 × 10 6 cells were washed with phosphate-buffered saline and stained with the following antibodies that were used for phenotypic analysis: CD3(APC-H7, SK-7) CD16 (PE-Cy7, B73.1), CD56 (APC R700, NCAM16.2), CD57 (BV605, NK-1), NKG2A (PE-Cy7, Z199), CD4 (APC-H7), CD8 (Per-CP Cy), CD45RA (FITC), and CD45RO (BV605), from BD Biosciences, (San Jose, CA, United States) and NKG2C (PE, REA205) from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Lipid Receptor GPR31 (G-Protein–Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis

doi: 10.1161/atvbaha.120.315154

Figure Lengend Snippet: Figure 6. GPR31 (G-protein–coupled receptor 31) regulates PAR (protease-activated receptor)-4–mediated platelet activation resulting in orchestrated control of Rap1-RASA3–mediated αIIbβ3 activation. A, Fura-2–labeled transiently transfected GPR31/Chinese hamster ovary (CHO) cells were stimulated with 10 and 20 μM 12(S)-HETE, and Ca2+ flux was measured over time. B, Fura-2–labeled human platelets were stimulated with 10 μM 12(S)-HETE, and Ca2+ flux was measured over time. C, GPR310 (G-protein–coupled receptor 310) blocks PAR4-mediated calcium flux on human platelets. Platelets were preincubated with 3 μM GPR310 or vehicle before stimulation with 160 μM AYPGKF. D, Immunoblot showing 3 nmol/L thrombin activation of pAKT or pERK phosphorylation after 15 min in human platelets pretreated with 1 μM GPR310 as indicated. β-actin is used as loading control. E, Gel-purified platelets were treated with GPR310 (3 μM), AZD1283 (10 μM), or dual inhibition before thrombin stimulation (3 nmol/L) for 15 min, and Rap1 activation was measured. The bottom panels represent total Rap1 as loading control. F, Human platelets were treated with GPR310 (1 μM), AZD1283 (10 μM), dual inhibition, or LY294002 (10 μM), before thrombin stimulation with 3 nmol/L thrombin for 5 min. After membrane preparation, samples were immunoblotted with RASA3 ab. β-actin is used as loading control. G, Schematic of the PAR4, GPR31, and P2Y12 signaling in human platelet. Molecular weight markers (kDa). 12-LOX indicates 12-lipoxygenase.

Article Snippet: For RASA3, platelet lysates were ultracentrifuged at 100K for 2 hours at 4 °C and membrane protein quantified using the Bradford assay, equally loaded and resolved by SDS-PAGE and Western blot and Rasa3 levels detected with Rasa3 Ab (Proteintech; No. 27835-1-AP).

Techniques: Activation Assay, Control, Labeling, Transfection, Western Blot, Phospho-proteomics, Purification, Inhibition, Membrane, Molecular Weight

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: IP5K binds CRL, generating IP6 to assemble CRL–CSN complexes in vivo. (A) IP5K pulls down CSN subunits and the cullins. (B) Coexpression of myc-Cul4A and GST-IP5K or its kinase-dead K138A mutant, followed by GST pulldown. (C) Coexpression of GST-IP5K and myc-Cul4A or its various N-terminal truncation mutants, followed by GST pulldown and myc immunoprecipitation. (D) Direct in vitro binding between recombinant IP5K and HEK293-purified Cul4A wild-type or the C-terminal domain (Δ400). (E) Inositol profiling of cells with shRNA-mediated knockdown of IP5K. (Inset) Western blotting of IP5K. (F) CSN1 immunoprecipitation in control and IP5K knockdown cells. (G) GST pulldown of Cul4A and Cul3 in control and IP5K knockdown cells. (H) IP6 addition to cell lysates increases binding between Cul4A and CSN in IP5K knockdown but not control cells. (I) Bacterial phytase treatment (10 µg/mL, 12 h, 4 °C) disrupts the CRL4A–CSN complex.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: In Vivo, Mutagenesis, Immunoprecipitation, In Vitro, Binding Assay, Recombinant, Purification, shRNA, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: IP5K binds CRL, generating IP6 to assemble CRL–CSN complexes in vivo. (A) GST-PPIP5K1/2 pulldown does not coprecipitate with CSN subunits or cullins. (B) GST-IP5K pulldown of Cul4A and CSN2 with/without UV radiation (100 J/m2). (C) Real-time PCR analysis of IP5K knockdown. PCR was performed as previously described (47). (D) shRNA-mediated knockdown of IP5K diminishes binding between CSN2 and Cul1/2. (E) IP6 (1 µM) increases Cul4A–CSN, but not Cul3–CSN, complex formation in IP5K knockdown cells. (F) PAGE imaging of IP6 after incubation with cell lysis buffer or cell lysate. (G) TNP (10 µM) increases Cul4A–CSN2 binding in wild-type but not IP5K knockdown cells. (H) Overexpressing IP6K increases Cul4A–CSN2 binding in IP5K knockdown cells, whereas further addition of TNP has no effect on Cul4A–CSN2 interactions in IP5K knockdown cells.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, shRNA, Binding Assay, Imaging, Incubation, Lysis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: IP5K regulates Cul1/4A neddylation and function. (A) Levels of neddylated cullins in control and shIP5K cells. Lysis buffer contains 1 mM N-ethylmaleimide to minimize deneddylation during processing. (B) Increased levels of K48-linkage–specific polyubiquitination of Cul1 and Cul4A in IP5K knockdown cells. (C) Levels of cullin substrates. (D) Rescue of shIP5K cells with wildtype or kinase-dead IP5K that are resistant to the shRNA because of synonymous mutations. (E) Growth curve of control and shIP5K cells. (F) IP5K depletion exacerbates UV-induced apoptosis, as measured by TUNEL assay. Cells were treated with UV (100 J/m2). After 20 h, cells were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated TUNEL assay system. Fragmented apoptotic cell nuclei were visualized by TUNEL (TdT, green), and the nucleus was stained with DAPI (blue). (G) Viability of control and shIP5K cells after treatment with MLN4924 (0.05, 0.25, 1, 2.5, 5 µM) for 48 h. (H) Scheme depicting the interconversion of cullins between a CSN-bound, inactive state and a CDC34-bound, active state. In this model, CSN2 and E2 ligase CDC34 both bind to cullins’ basic canyon via electrostatic interactions (23). IP6 generated by IP5K mediates high-affinity, inactive cullin–CSN complexes, whereas IP6K1 metabolizes IP6 leading to decreased complex affinity (21), which may be followed by cullin activation and CDC34 binding.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: Lysis, shRNA, TUNEL Assay, Staining, Generated, Activation Assay, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: IP5K regulates Cul1/4A neddylation and function. (A) Levels of neddylated cullins in control and shIP5K (#2) cells. (B) Levels of neddylated cullins with/without MG132 (10 µM) and MLN4924 (0.2 µM) treatments (4 h).

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: