Journal: Scientific Reports

Article Title: Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFκB pathways

doi: 10.1038/srep04905

Figure Lengend Snippet: (A–C), THP-1 cells were incubated with OA-NO2 and 1-LG at the indicated concentrations for 1 h and then stimulated with PMA (0.05 μM) and LPS (1 μg/ml) for 24 h. The release of proinflammatory cytokines (IL6, MCP-1 and TNFα) into the medium was determined by ELISA. (D), Both shRNA-Lp-PLA2 and a negative shRNA control were transfected into THP-1 cells for 24 h prior to stimulation with 0.05 μM PMA for another 24 h. (E and F), The down-regulation of the expression of Lp-PLA2 by shRNA. (G), An Lp-PLA2 overexpression plasmid and pcDNA3.1 plasmid were transfected into 293T cells for 24 h, and the medium were used to treat THP-1 cells together with PMA. Then, SOD activity was determined. (H), The PAF-AH activity in the medium of the transfected 293T cells.

Article Snippet: The levels of proinflammatory cytokines in the culture media were measured with a Human IL-6 ELISA Kit, a Human TNFα ELISA Kit and a Human MCP-1 ELISA Kit (Boster Inc., China), following the manufacturer's instructions.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, shRNA, Control, Transfection, Expressing, Over Expression, Plasmid Preparation, Activity Assay

Journal: Journal of cellular biochemistry

Article Title: Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

doi: 10.1002/jcb.29903

Figure Lengend Snippet: FIGURE 5 Combination of GGA and rM180 stimulation upregulates IL‐8, MCP‐1, and IL‐6 production in hPDLCs. (A) Heat‐map for the comparison of the gene expression between hPDLCs stimulated with GGA for 18 h and those that were stimulated with GGA for 18 h, followed by rM180‐stimulation for 6 h. Each row represents a gene and each column represents a sample that was treated as indicated. Red and green blocks represent a higher and lower expression relative to the control sample (unstimulated 0 h), respectively. Data on the genes whose expression was increased in the GGA stimulation group than that in the unstimulated group were extracted with a ratio of 4.0 or higher. (B–J) hPDLCs were stimulated with or without GGA, rM180, or GGA + rM180 for 21, 30, or 42 h. Culture supernatants were collected, and the secretion of IL‐8, MCP‐1, and IL‐6 was quantified by using an ELISA sandwich assay. Data are represented as mean ± SD. n = 3. ELISA, enzyme‐linked immunosorbent assay; GGA, geranylgeranylacetone; hPDLC, human periodontal ligament cell; IL‐8, interleukin‐8; MCP‐1, monocyte chemotactic protein 1. *p < .05; **p < .01; ***p < .001

Article Snippet: The concentration of interleukin‐8 (IL‐8), monocyte chemotactic protein 1 (MCP‐1), and IL‐6 in the supernatant samples was measured using the corresponding ELISA kit (Proteintech).

Techniques: Comparison, Gene Expression, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer

doi: 10.1038/s41419-019-1656-4

Figure Lengend Snippet: a ELISA analysis of SCF levels in ascitic effusions collected from EOC patients ( N = 32). b ELISA analysis of SCF performed on conditioned media of FACS-sorted CD44 + c-Kit + (CSC) and CD44 + c-Kit − (no-CSC) cells from primary samples of EOC ascitic effusions. Raji cells and ascites from EOC patients were used as negative and positive control, respectively. The bars represent the mean ± S.D. ( N = 4). N.D. = not detectable. Sorting gating strategy is shown on the left. c Flow cytometry analysis of SCF on CSC and no-CSC from primary samples of EOC ascitic effusions. The bars represent the mean ± S.D. ( N = 4). Gating strategy is shown on the left. d qRT-PCR analysis of SCF 248 and SCF 220 isoforms in FACS-sorted CSC and no-CSC from EOC ascitic effusions. Data were normalized to tumor infiltrating lymphocytes (TIL). The bars represent the mean ± S.D. ( N = 4). Schematic representation of SCF isoforms is shown. e ELISA analysis of SCF on conditioned media of TAF, TAM and TIL sorted by FACS from primary samples of EOC ascitic effusions. Data are expressed as mean ± S.D. ( N = 4). N.D. = not detectable. f Flow cytometry analysis of SCF on TAF, TAM, and TIL from primary samples of EOC ascitic effusions. The bars represent the mean ± S.D. ( N = 4). Gating strategy is shown. g qRT-PCR analysis of SCF 248 and SCF 220 isoforms in FACS-sorted TAF, TAM, and TIL from EOC ascitic effusions. Data were normalized to TIL. The bars represent the mean ± S.D. ( N = 4). h Schematic representation of SCF isoform expression in the different cell subsets within the EOC microenvironment

Article Snippet: For membrane-associated SCF overexpression, pLenti-C-mGFP-P2A-Puro plasmid encoding GFP-tagged human KIT ligand, transcript variant “a”, as well as the empty vector, were purchased from OriGene Technologies (Rockville, MD).

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Flow Cytometry, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer

doi: 10.1038/s41419-019-1656-4

Figure Lengend Snippet: a WB analysis of p-c-Kit and p-Akt in Kasumi-1 cells pretreated or not with imatinib (5, 10, 30, and 50 μM) and stimulated with hrSCF (50 ng/mL) for 5 min. Signals were normalized to β-actin. A representative blot is shown. The bars represent p-c-Kit/c-Kit and p-Akt/Akt mean ± S.D. ( N = 3). *, # p < 0.05 for p-Akt, and p-c-Kit, respectively. b Flow cytometry analysis of p-Akt in Kasumi-1 cells pre-treated or not with imatinib (30 μM) and stimulated with hrSCF (50 ng/mL) for 5 min. Representative FACS plots are shown. The bars represent the mean ± S.D. ( N = 3). c WB analysis of p-c-Kit and p-Akt in Kasumi-1 cells pretreated or not with imatinib (30 μM) and stimulated with hrSCF (50 ng/mL) or hrSCF-coated wells for 5 min. Signals were normalized to β-actin. A representative blot out of 3 independent experiments is shown. d Flow cytometry analysis of p-Akt in Kasumi-1 cells pretreated or not with 30 μM imatinib and co-cultured for 5 min with Raji-CTRL or Raji overexpressing SCF (Raji-SCF). Representative FACS plots are shown. The bars represent the mean ± S.D. ( N = 3). ** p < 0.01; *** p < 0.001

Article Snippet: For membrane-associated SCF overexpression, pLenti-C-mGFP-P2A-Puro plasmid encoding GFP-tagged human KIT ligand, transcript variant “a”, as well as the empty vector, were purchased from OriGene Technologies (Rockville, MD).

Techniques: Flow Cytometry, Cell Culture

Journal: Cell Death & Disease

Article Title: A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer

doi: 10.1038/s41419-019-1656-4

Figure Lengend Snippet: a Representative WB analysis of c-Kit and p-c-Kit expression in EOC cells cultured either under adherent or spheroid-forming conditions. Kasumi-1 cells stimulated for 5 min with SCF were used as positive control for p-c-Kit (ctrl + ). The bars represent the mean ± S.D. ( N = 4). N.D. = not detectable. b WB analysis of p-Akt in EOC cells cultured under spheroid-forming conditions pretreated or not with imatinib (30 μM) and stimulated with hrSCF (50 ng/mL) for 5 min. Signals were normalized to β-actin. A representative blot is shown. The bars represent p-Akt/Akt mean ± S.D. ( N = 3). c ELDA performed on EOC cells cultured for two weeks under spheroid-forming conditions in the presence of hrSCF (50 ng/mL), imatinib (5 μM) or the combination of the two. The bars represent the mean ± S.D. ( N = 3). d Representative WB analysis of PDGFR-α and β in EOC samples. NIH3T3 were used as positive control. e qRT-PCR analysis of Oct4, Sox2, Nanog, and c-Kit mRNA levels in EOC cells cultured for two weeks as described in a . The bars represent the mean ± S.D. ( N = 3). * p < 0.05, ** p < 0.01

Article Snippet: For membrane-associated SCF overexpression, pLenti-C-mGFP-P2A-Puro plasmid encoding GFP-tagged human KIT ligand, transcript variant “a”, as well as the empty vector, were purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Cell Culture, Positive Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs

doi: 10.1038/s41467-020-16616-8

Figure Lengend Snippet: a TBC1D15 knockdown inhibits NUMB phosphorylation at S265 as demonstrated by immunoblot analysis. b TBC1D15 knockdown or overexpression does not affect kinase protein levels as demonstrated by immunoblot analysis. c TBC1D15 knockdown reduces and TBC1D15 overexpression increases aPKCζ kinase activity demonstrated aby in vitro kinase assays. Data are represented as ±SD ( n = 4). p -Values by two-tailed unpaired t test. * p = 0.00027596 (shTBC1D15 vs shScramble), * p = 0.000008157 (shScramble vs TBC1D15 OE) (Student’s t test). d In vitro cell-free NUMB phosphorylation assay. Each bar represents the mean ± SD of four independent experiments. p -Values by two-tailed unpaired t test. * p = 0.000139 (TBC1D15–AURKA–aPKCζ-NUMB vs AURKA–aPKCζ-NUMB), * p = 0.0002468 (TBC1D15–AURKA–aPKCζ-NUMB vs TBC1D15-aPKCζ-NUMB), * p = 0.00000058 (TBC1D15–AURKA–aPKCζ-NUMB vs TBC1D15–AURKA–aPKCζ-NUMB-0.25μg RANGAP1), * p = 0.00008243 (TBC1D15–AURKA–aPKCζ-NUMB vs TBC1D15–AURKA–aPKCζ-NUMB-0.5 μg RANGAP1), * p = 0.0007145 (TBC1D15–AURKA–aPKCζ-NUMB-shScramble vs TBC1D15–AURKA–aPKCζ-NUMB-shRANGAP1) (Student’s t test). e (top) RANGAP1 knockdown abrogated and RANGAP1 overexpression (OE) enhances AURKA interaction with aPKCζ and TBC1D15 in Huh7 cells as determined by co-IP-Western blot analysis. (bottom) Immunoblot of total cell lysate reveals p-NUMB was reduced by RANGAP1 KD and increased by RANGAP1 OE. PAR6. f We tested if serine or threonine residue of NuMA1 is phosphorylated by Aurora-A, and that this phosphorylation is important for association with TBC1D15. Serine or threonine residue of NuMA1 is phosphorylated by Aurora-A. g Phosphorylation of serine or threonine residue of NuMA1 by Aurora-A is important for asymmetric cell division, judged by immunofluorescence staining of α-Tubulin and NuMA1-GFP intensity. Bar graph represents fold enrichment of GFP-NuMA1. Scale bar, 10 μm. Data are represented as ±SD ( n = 3). p -Values by two-tailed paired t test. * p = 0.000667 (WT vs T1991A) * p = 0.000936 (WT vs S2047A) (Student’s t test). h Hypothetical model that TBC1D15 expression promotes NUMB phosphorylation via RANGAP1–TBC1D15 interaction enhancing AURKA–aPKCζ interaction and activation. Source data are provided as a file.

Article Snippet: Aurora A and aPKCζ were measured by SignalChem Kinase activity assay Kits as described previously.

Techniques: Western Blot, Over Expression, Activity Assay, In Vitro, Two Tailed Test, Phosphorylation Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Activation Assay

Journal: iScience

Article Title: Sebocytes contribute to melasma onset

doi: 10.1016/j.isci.2022.103871

Figure Lengend Snippet: Effect of UVA irradiation on growth factors release in SZ95 sebocytes (A) Experimental scheme of UVA irradiation of SZ95 sebocytes. (B) Phase-contrast analysis of SZ95 sebocytes after 4-UVA 5 J/cm 2 irradiations. (C) The mRNA expression levels of POMC, EDN1, SCF, and b-FGF in SZ95 sebocytes after 24 and 48h post 1-UVA and 48h post three or 4-UVA 5 J/cm 2 irradiations. Results are presented as the mean ± SD of three independent experiments and are expressed as the fold change respect to untreated control cells (∗p < 0.05, ∗∗p < 0.01). (D) Protein quantitation by ELISA of α-MSH, EDN1, SCF, and b-FGF in SZ95 sebocytes after 24 and 48h post 1-UVA and 48h post three or 4-UVA 5 J/cm 2 irradiations. Results are presented as the mean ± SD of three independent experiments and are expressed in absolute quantities (∗p < 0.05, ∗∗p < 0.01 vs Ctr). (E) The mRNA expression levels and protein quantitation by ELISA of POMC/α-MSH, EDN1, SCF, and b-FGF in SZ95 sebocytes after 72h or 1 week post 7-UVA 5 J/cm 2 irradiations. Data represent the mean ± SD of three independent experiments. For mRNA levels, results are expressed as the fold change respect to untreated control cells (∗∗p < 0.01). For ELISA assay, results are expressed in the absolute quantities (∗∗p < 0.01 vs Ctr).

Article Snippet: Human SCF ELISA Kit , Origene Technologies Inc. , Cat#EA102189.

Techniques: Irradiation, Expressing, Control, Protein Quantitation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sebocytes contribute to melasma onset

doi: 10.1016/j.isci.2022.103871

Figure Lengend Snippet: Effect of UVA irradiation on inflammatory mediators release in SZ95 sebocytes (A) The mRNA expression levels of IL-1α, IL-1β, IL-6, IL-8, and protein quantitation by ELISA of IL-6 and IL-8 in SZ95 sebocytes after 24 and 48h post 1-UVA and 48h post three or 4-UVA 5 J/cm 2 irradiations. For mRNA levels, results are expressed as the fold change respect to untreated control cells (∗p < 0.05, ∗∗p < 0.01). For ELISA assay, results are expressed in absolute quantities (∗p < 0.05, ∗∗p < 0.01 vs Ctr). (B) PGD2, PGE2, PGF2α, LTB4, and AA quantitation by HPLC-MS/MS in SZ95 sebocytes after 24 and 48h post 1-UVA and 48h post three or 4-UVA 5 J/cm 2 irradiations. Results are expressed in absolute quantities (∗p < 0.05, ∗∗p < 0.01 vs Ctr). Data represent the mean ± SD of three independent experiments.

Article Snippet: Human SCF ELISA Kit , Origene Technologies Inc. , Cat#EA102189.

Techniques: Irradiation, Expressing, Protein Quantitation, Enzyme-linked Immunosorbent Assay, Control, Quantitation Assay, Tandem Mass Spectroscopy

Journal: iScience

Article Title: Sebocytes contribute to melasma onset

doi: 10.1016/j.isci.2022.103871

Figure Lengend Snippet: Effect of UVA irradiation on p38MAP kinase and p53 signaling in SZ95 sebocytes (A) Western blot analysis of phospho-p38 protein expression in SZ95 sebocytes after 1-2-4h post irradiation with UVA 2-5-8 J/cm 2 β-tubulin was used as an equal loading control. Representative blots are shown. Densitometric scanning of band intensities was performed to quantify the change of protein expression (control value taken as 1-fold in each case). (B) Western blot analysis of p53 and p21 protein expression in SZ95 sebocytes after 24 and 48h post 1-UVA and 48h post three or 4-UVA 2-5-8 J/cm 2 . GAPDH was used as an equal loading control. Representative blots are shown. Densitometric scanning of band intensities was performed to quantify the change of protein expression (control value taken as 1-fold in each case). (C) The mRNA expression levels of POMC, EDN1, SCF, and b-FGF in SZ95 sebocytes after 48h of treatment with PFTα 5 μM and one or 3-UVA 5 J/cm 2 irradiations. Results are presented as the mean ± SD of three independent experiments and are expressed as the fold change respect to untreated control cells (∗p < 0.05, ∗∗p < 0.01 vs Ctr; $ p < 0.05 vs 1-UVA irradiated cells; § p < 0.05 vs 3-UVA irradiated cells). (D) Protein quantitation by ELISA of αMSH, EDN1, SCF, and b-FGF in SZ95 sebocytes after 48h of treatment with PFTα 5 μM and one or 3-UVA 5 J/cm 2 irradiations. Results are presented as the mean ± SD of three independent experiments and are expressed in absolute quantities (∗∗p < 0.01 vs Ctr; $ p < 0.05 vs 1-UVA irradiated cells; § p < 0.05 vs 3-UVA irradiated cells).

Article Snippet: Human SCF ELISA Kit , Origene Technologies Inc. , Cat#EA102189.

Techniques: Irradiation, Western Blot, Expressing, Control, Protein Quantitation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sebocytes contribute to melasma onset

doi: 10.1016/j.isci.2022.103871

Figure Lengend Snippet: Melanogenesis promotion and growth factors induction in e x vivo and in vivo systems (A) Experimental schemes and macroscopic visualization of ex vivo skin explants coculture with UVA irradiated SZ95 sebocytes. (B) Experimental schemes and macroscopic visualization of ex vivo skin explants cultured with conditioned medium from irradiated SZ95 sebocytes. (C and D) Pigmentation of ex vivo skin explants visualized by Fontana-Masson staining. Scale bars: 50 and 20 μM for low and high magnification, respectively. (E and F) Immunohistochemical analysis of SCF expression. Scale bars: 50 and 20 μM for low and high magnification, respectively. (G) Immunohistochemical analysis of SCF expression in the sebaceous glands of ex vivo skin explants stimulated with conditioned medium from control and irradiated SZ95 sebocytes. Scale bars: 50 μM (H) Western blot analysis of p53 protein expression on sebutape from lesional (L) or non-lesional (NL) melasma skin. GAPDH was used as an equal loading control. Representative blots are shown. Densitometric scanning of band intensities was performed to quantify the change of protein expression (NL control value taken as 1-fold in each case). (I) Protein quantitation by ELISA of α-MSH, EDN1, SCF, and b-FGF in sebum samples. Results are presented as the mean ± SD and are expressed in absolute quantities (∗p < 0.05 vs Ctr).

Article Snippet: Human SCF ELISA Kit , Origene Technologies Inc. , Cat#EA102189.

Techniques: In Vivo, Ex Vivo, Irradiation, Cell Culture, Staining, Immunohistochemical staining, Expressing, Control, Western Blot, Protein Quantitation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sebocytes contribute to melasma onset

doi: 10.1016/j.isci.2022.103871

Figure Lengend Snippet:

Article Snippet: Human SCF ELISA Kit , Origene Technologies Inc. , Cat#EA102189.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Ex Vivo, Software, Modification