Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: Loss of Shp-2 leads to increased mature NK cell numbers. a , b Representative flow cytometric image, frequency, and number of splenic NK cells (gated as NK1.1 + CD3/CD19 − ) of Ncr1 Ki Ptpn11 fl/fl (dark gray) and control mice (white) are depicted ( a ) or of splenic NK cells (gated as NK1.1 + CD3 − YFP + NKp46 + ) of Ncr1 Tg Ptpn11 fl/fl (light gray) or Ncr1 Tg Ptpn11 wt/fl (white) mice ( b ) are depicted. c , d Analysis of NK cells from Ncr1 Ki Ptpn11 fl/fl and control mice. c Representative cytometric plot of BM NK cells (gated as CD122 + NK1.1 + NKp46 + CD3/CD19 − ) stained with CD27 and CD11b; percentage and number of CD27 single-positive (CD27 SP; CD27 + CD11b − ), double-positive (DP; CD27 + CD11b + ), and CD11b single-positive (CD11b SP; CD27 − CD11b + ) populations are shown. d Representative cytometric plot of splenic NK cells (gated as NK1.1 + NKp46 + CD3/CD19 − ) stained with CD27 and CD11b; percentage and number of the afore-mentioned subsets are shown. e As in ( d ), but for NK cells gated as NK1.1 + CD3 − YFP + NKp46 + from Ncr1 Tg Ptpn11 fl/fl or Ncr1 Tg Ptpn11 wt/fl mice. f Frequency and number of CD27 SP, DP, and CD11b SP conventional NK cells (gated as CD45 + NK1.1 + CD3 − YFP + NKp46 + DX5 + CD49a − ) from Ncr1 Tg Ptpn11 fl/fl and control mice in the liver and a representative staining of CD27 and CD11b expression. a , b Results represent the mean ± SEM of n = 8 ( Ncr1 Ki Ptpn11 fl/fl ) and n = 13 ( Ncr1 Ki Ptpn11 wt/wt ) ( a ), n = 29 ( Ncr1 Tg Ptpn11 wt/fl ) and n = 35 ( Ncr1 Tg Ptpn11 fl/fl ) ( b ) mice per genotype, are a pool of three ( a ) and nine ( b ) experiments, which are representative of at least five ( a ) and eleven ( b ) independent experiments. c – f Results represent mean ± SEM of n = 4 ( Ncr1 Ki Ptpn11 fl/fl ) and n = 6 ( Ncr1 Ki Ptpn11 wt/wt ) ( c ), n = 7 ( Ncr1 Ki Ptpn11 fl/fl ) and n = 10 ( Ncr1 Ki Ptpn11 wt/wt ) ( d ), n = 29 ( Ncr1 Tg Ptpn11 wt/fl ) and n = 35 ( Ncr1 Tg Ptpn11 fl/fl ) ( e ), and n = 16 ( Ncr1 Tg Ptpn11 wt/fl ) and n = 20 ( Ncr1 Tg Ptpn11 fl/fl ) ( f ) mice per genotype, are a pool of two ( d ), nine ( e , f ) experiments and representative of at least three ( c , d ), and eleven ( e , f ) independent experiments. Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Staining, Expressing

Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: Shp-2 is largely dispensable for NK cell effector functions. a Graphs depict percentages of CD94 + , Ly49A + , Ly49G2 + , Ly49I + , Ly49D + , Ly49H + , and KLRG1 + splenic NK cells (gated as NK1.1 + CD3/CD19 − ) from Ncr1 Ki Ptpn11 wt/wt (white), Ncr1 Ki Ptpn11 fl/fl (dark gray), and B2m −/− (green) mice. b Graph and a representative cytometric plot illustrate the production of granzyme A and B by splenic NK cells (gated as NK1.1 + NKp46 + CD3/CD19 − ) after phorbol 12-myristate 13-acetate and ionomycin stimulation (PMA/Iono). c NK cells isolated from polyinosinic:polycytidylic acid (polyI:C)-treated Ncr1 Ki Ptpn11 wt/wt or Ncr1 Ki Ptpn11 fl/fl mice were plated with RMA, RMA-S, or RMA-H60 cells at the indicated ratios. The graph depicts percentage killing of target cells, as measured by quantifying PI − living target cells. d Naive splenocytes from Ncr1 Tg Ptpn11 fl/fl (light gray) mice or heterozygote littermate controls (white) were incubated with RMA cells or RMA-m157 cells (expression of m157 is illustrated in the graph on the left). Percentage of YFP + Ly49H + CD107α + NK cells in each group was determined by flow cytometry (illustrated in the graph on the right). a , b Results represent the mean ± SEM of n = 4 ( Ncr1 Ki Ptpn11 fl/fl or B2m −/− ) and n = 6 ( Ncr1 Ki Ptpn11 wt/wt ) mice per genotype and are representative of at least three ( a ) and two ( b ) independent experiments. c , d Results represent the mean ± SD of n = 3 ( c ), n = 4 (for non-stimulated conditions) and n = 5 (for RMA conditions) ( d ) technical replicates and are representative of at least two ( c ) and three ( d ) independent experiments. Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Isolation, Incubation, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: Ptpn11 -deficient NK cells show a biphasic response to IL-15. a – d Enriched NK cells from Ncr1 Ki Ptpn11 wt/wt (blue; congenically marked) and Ncr1 Ki Ptpn11 fl/fl (red) mice were co-cultured for 5 days in the presence of the indicated amounts of IL-15 ( a , b ) or were co-cultured for 4 days with 50 ng/ml of IL-2 ( c , d ). a , c Histograms represent the amount and cell division (CTV dilution) of Shp-2-deficient and control NK living cells in each condition (quantitative flow cytometry acquisition) and ratios thereof. b , d Representative flow cytometry plots show the percentage of dead cells (PI + ) among knockout and control NK cells and ratios thereof. e Enriched Shp-2-deficient and congenically marked control NK cells were co-cultured for 4 days in the presence of the indicated amounts of IL-15 and 0 (black triangles) or 20 nM ABT-199 (pink triangles). The graph depicts the ratio of living knockout over control NK cells. f Enriched NK cells from C57BL/6 wild-type mice (blue), or Shp-2-deficient NK cells from Ncr1 Ki Ptpn11 fl/fl mice (red) as control, were cultured for 4 days in the presence of 0.1 or 50 ng/ml IL-15 and 0, 0.25, 0.5, or 1 μM SHP099. Graphs depict the number of living (PI − ) NK cells. Results represent the mean ± SD of n = 3 ( a , b ) and n = 4 ( c , d , f ) replicates, or of n = 3 independent experiments ( e ; average of each condition/experiment). Results are representative of at least five ( a , c ), two ( b , d ), or three ( e , f ) independent experiments. Statistical comparisons are shown in ( e ), ( f ); NS, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; Student’s t -test unpaired ( f ) and Student’s t -test paired ( e ). Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Cell Culture, Flow Cytometry, Knock-Out

Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: Shp-2-deficiency alters NK cell size and granularity. a , b Enriched NK cells from Ncr1 Ki Ptpn11 wt/wt (blue; congenically marked) and Ncr1 Ki Ptpn11 fl/fl (red) mice were co-cultured for 5 days in the presence of the indicated amounts of IL-15. a Quantification of the geometric mean of forward scatter (FSC) and side scatter (SSC) of knockout and control NK cells (the average of the NK cells in culture with 0.1 ng/mL of IL-15 from Ncr1 Ki Ptpn11 wt/wt mice was set as 100%). b Quantification and representative flow cytometry plots depicting the geometric mean of FSC and SSC of non-divided and divided NK cells (the average of the non-divided NK cells from Ncr1 Ki Ptpn11 wt/wt mice was set as 100%). c , d Enriched NK cells from Ncr1 Ki Ptpn11 wt/wt (white) and Ncr1 Ki Ptpn11 fl/fl (dark gray) mice were co-cultured for 2 days in the presence of 50 ng/ml of IL-15. Representative flow cytometry plots illustrating Ki67 and SSC and a quantification of the percentage of SSC high cells ( c ) and of Ki67 + among SSC high ( d ) non-divided control and knockout NK cells (gated as NK1.1 + CD3/CD19 − and cell trace violet (CTV) high). e , f Representative cytometric profile and quantification of FSC and SSC for total ( e ), CD27 SP, DP, and CD11b SP NK cell populations ( f ) in the spleen (gated as NK1.1 + NKp46 + CD3/CD19 − ). For quantification, the average of Ncr1 Ki Ptpn11 wt/wt was set as 100%. Results represent the mean ± SD of n = 3 ( a , b ) and n = 10 ( c , d ) technical replicates per genotype and are representative of at least two independent experiments ( a , b ) or are a pool of two independent experiments ( c , d ). Results represent the mean ± SEM of n = 10 ( Ncr1 Ki Ptpn11 fl/fl ) and n = 13 ( Ncr1 Ki Ptpn11 wt/wt ) mice ( e , f ) per genotype and are a pool of three independent experiments ( e , f ). Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Cell Culture, Knock-Out, Flow Cytometry

Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: Ptpn11 -deficient NK cells have a curtailed response to IL-15. a Schematic representation of the main signaling pathways engaged downstream of IL-15R in NK cells. b , c Purified NK cells ( b ) or splenocytes ( c ) from Ncr1 Ki Ptpn11 wt/wt and Ncr1 Ki Ptpn11 fl/fl mice were cultured in vitro for 30 min in the presence of 50 ng/ml IL-15 (blue and red, respectively) or left untreated (white and dark gray, respectively). Phosphorylation of STAT5, ERK, and S6 were measured by immunoblot analysis ( b ) and by flow cytometry in CD27 SP, DP, and CD11b SP NK cell subsets (gated as NK1.1 + and CD3/CD19 − ) ( c ). d Purified NK cells from C57BL/6 wild-type mice were cultured in vitro for 30 min in the presence of 50 ng/ml IL-15 or left untreated and in the presence of 4 μM SHP099. Phosphorylation of the indicated proteins was measured by immunoblot analysis. Actin was used as loading control ( b , d ). e Phosphorylation of ERK and S6 (expressed as geometric MFI) on splenic NK cells of untreated mice or of mice treated with IL-15/IL-15R complexes 1 day prior to the analysis are illustrated. Results represent the mean ± SEM of n = 5 ( Ncr1 Ki Ptpn11 wt/wt ) and n = 6 ( Ncr1 Ki Ptpn11 fl/fl ) ( c ), n = 6 ( Ncr1 Ki Ptpn11 fl/fl ) and n = 7 or n = 8 ( Ncr1 Ki Ptpn11 wt/wt ) ( e ) mice per genotype and are a pool of two independent experiments ( e ) or representative of at least two independent experiments ( b – d ). Statistical comparisons are shown ( c , e ); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Purification, Cell Culture, In Vitro, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: mTOR and ERK inhibition phenocopy the expansion defect of Shp-2-deficient NK cells. a Splenocytes from C57BL/6 wild-type mice were cultured in vitro for 30 min in the presence of 50 ng/ml IL-15 (blue) and in the presence of 50 µM PD98059 or 250 nM Torin2, or left untreated (white). Phosphorylation of the indicated proteins was measured by flow cytometry. b Splenocytes from Ncr1 Ki Ptpn11 wt/wt and Ncr1 Ki Ptpn11 fl/fl mice were cultured in vitro for 30 min in the presence of 50 ng/ml IL-15 (blue and red, respectively) or left untreated (white and dark gray, respectively). Phosphorylation of Akt (position 308) was measured by flow cytometry in CD27 SP, DP, and CD11b SP NK cell subsets (gated as NK1.1 + and CD3/CD19 − ). For quantification, the average of the untreated NK cells from Ncr1 Ki Ptpn11 wt/wt mice was set as 100%. c , d NK cells from C57BL/6 wild-type mice were cultured in vitro with 50 ng/ml IL-15 for 4 days; PD98059 was added daily (day 0, 1, 2, and 3) at a concentration of 25 μM, while Torin2 was added once (day 0) at a concentration of 5 nM. c Histograms represent the amount and cell division (CTV dilution) of living NK cells in each condition (quantitative flow cytometry acquisition) and graph the number of living NK cells (gated as propidium iodide (PI) – NK1.1 + CD3/CD19 – ) after 4 days of culture for each condition. d Graphs depict the geometric mean of FSC and SSC of non-divided NK cells across conditions (gated as PI – NK1.1 + CD3/CD19 − and cell trace violet (CTV) high). For quantification, the average of IL-15-expanded NK cells was set as 100%. Results represent the mean ± SEM of n = 9 ( a ), n = 9 ( Ncr1 Ki Ptpn11 wt/wt ) and n = 10 mice ( Ncr1 Ki Ptpn11 fl/fl ) ( b ) and are a pool of two independent experiments ( a , b ). Results represent the mean ± SD of n = 5 technical replicates and are representative of two independent experiments ( c , d ). Statistical comparisons are shown ( a – d ); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Inhibition, Cell Culture, In Vitro, Flow Cytometry, Concentration Assay

Journal: Nature Communications

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

doi: 10.1038/s41467-019-09431-3

Figure Lengend Snippet: Shp-2 mediates NK cell activation in response to IL-15 in vivo. a , b CPD-labeled splenic YFP + NK cells from Ncr1 Tg Ptpn11 fl/fl (CD45.2 + ; light gray line and bars) mice were sorted and injected at a 1:1 ratio with sorted splenic NK cells from B6.SJL (CD45.1 + ; black line and white bars) congenic mice into recipient Rag2 −/− Il2Rg −/− mice. Percentage of cells in divisions 1–3 or 4–7 ( a ) and relative proportion of total NK cells were determined at day 4 post-transfer ( b ). c , d Splenic YFP + NK cells from Ncr1 Tg Ptpn11 fl/fl ROSA EYFP (CD45.2 + ) mice were sorted and injected at a 1:1 ratio with sorted splenic NK cells from B6.SJL (CD45.1 + ) congenic mice into MCMV infected NK cell-deficient mice ( Ncr1cre Ki ROSA-DTA). Frequencies of total NK cells ( c ), and of Ly49H + and Ly49H − NK cells ( d ) were determined at day 7 post-infection. Results represent mean ± SEM of n = 3 mice ( a , b ) or of n = 13 mice ( c , d ), and are representative of three independent experiments ( a , b ) or a pool of three independent experiments ( c , d ). Statistical comparisons are shown; *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Article Snippet: NK cells were then grown in RPMI 1640 (Life technologies/Cat number 61870010) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (all from Life technologies), and 10 mM HEPES buffer (Bioconcept) and incubated at 37 °C in 5% CO 2 with recombinant mouse IL-15 PeproTech/Cat number 210-15) or IL-2 (PeproTech/Cat number 212-12) for 4 or 5 days in the presence or not of Bcl-2 inhibitor (ABT-199 from BioVision), Mcl-1 inhibitor (S63845 from Selleckchem), Shp-2 inhibitor (SHP099 from Selleckchem), the MEK (MAP kinase kinase) inhibitor (PD 98059 from Adipogen), or the mTORC1 inhibitor (Torin2 from Selleckchem) as indicated.

Techniques: Activation Assay, In Vivo, Labeling, Injection, Infection

Journal: International Journal of Molecular Sciences

Article Title: Activation of cAMP Signaling in Response to α-Phellandrene Promotes Vascular Endothelial Growth Factor Levels and Proliferation in Human Dermal Papilla Cells

doi: 10.3390/ijms23168959

Figure Lengend Snippet: α-phellandrene-mediated cyclic adenosine monophosphate (cAMP) accumulation stimulates the downstream protein kinase (PKA)/cAMP-responsive element-binding protein (CREB) signaling in dermal papilla cells (DPCs). DPCs were treated with vehicle (dimethyl sulfoxide; DMSO) or 12.5 μM α-phellandrene for 0.5, 1, and 3 h, following which ( A ) protein kinase A catalytic subunit (PKA Cα) expression and ( B ) CREB phosphorylation were quantified by western blotting. Cells were incubated with either vehicle or 12.5 μM of α-phellandrene for 0.5 h. The cells were preincubated with the vehicle or 50 μM of SQ22,536 (cAMP inhibitor) for 1 h before α-phellandrene treatment. ( C ) PKA Cα expression and ( D ) CREB phosphorylation were quantified by western blotting. Relative PKA Cα protein expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level and the ratio of CREB phosphorylation normalized to total CREB is shown ( p -CREB/CREB). Results are expressed as mean ± standard error of the mean (SEM) of three independent experiments. Statistically significant differences are marked as ns, not significant ( p > 0.05); * p < 0.05; ** p < 0.01.

Article Snippet: To investigate the mechanism underlying the proliferative effect of α-phellandrene, adenylyl cyclase inhibitor SQ22,536 (50 μM, Sigma-Aldrich), PKA inhibitor H89 (10 µM, Sigma-Aldrich), or CREB inhibitor 666-15 (100 nM; Selleckchem; Houston, TX, USA) was added 1 h before 12.5 μM of α-phellandrene treatment.

Techniques: Binding Assay, Expressing, Western Blot, Incubation