Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Skeletal inflammation and attenuation of Wnt signaling, Wnt ligand expression, and bone formation in atherosclerotic ApoE-null mice

doi: 10.1152/ajpendo.00501.2015

Figure Lengend Snippet: TaqMan reagents used for quantification of mRNA by qPCR

Article Snippet: Transcript levels were calculated by normalizing to the housekeeping gene mitochondrial ribosomal protein S2 using the ΔC T method ( 34 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 NCBI Gene Name Gene Name TaqMan # Alox12 Arachidonate 12-lipoxygenase Mm00545833_m1 Alox15 Arachidonate 15-lipoxygenase Mm01250458_m1 Axin2 Axin2 Mm00443610_m1 Mrps2 Mitochondrial ribosomal protein S2 (chob) Mm01962382_s91 Cx43 Gap junction membrane channel protein alpha 1 Mm01962382_s53 Dkk1 Dickkopf homolog 1 Mm00438422_m1 IL-1ß Interleukin-1β Mm00434228_m1 IL-6 Interleukin-6 Mm00446190_m1 Nkd2 Naked cuticle 2 homolog Mm00472240_m1 Sost Sclerostin Mm00470479_m1 Sfrp4 Secreted frizzled-related protein 4 Mm00840104_m1 TNF Tumor necrosis factor-α Mm00443258_m1 Tnfrsf11b Tumor necrosis factor receptor superfamily, member 11b (osteoprotogerin, OPG) Mm00435452_m1 Wisp1 WNT1 inducible signaling pathway protein 1 Mm00457574_m1 Wnt4 Wingless-related MMTV integration site 4 Mm00437341_m1 Wnt5a Wingless-related MMTV integration site 5A Mm00437347_m1 Wnt6 Wingless-related MMTV integration site 6 Mm00437353_m1 Wnt10b Wingless-related MMTV integration site 10B Mm00442104_m1 Wnt16 Wingless-related MMTV integration site 16 Mm00446420_m1 Open in a separate window TaqMan reagents used for quantification of mRNA by qPCR Statistics.

Techniques: Membrane

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Skeletal inflammation and attenuation of Wnt signaling, Wnt ligand expression, and bone formation in atherosclerotic ApoE-null mice

doi: 10.1152/ajpendo.00501.2015

Figure Lengend Snippet: Effect of HFD on expression of Wnt target genes, Wnt ligands, and Wnt antagonists

Article Snippet: Transcript levels were calculated by normalizing to the housekeeping gene mitochondrial ribosomal protein S2 using the ΔC T method ( 34 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 NCBI Gene Name Gene Name TaqMan # Alox12 Arachidonate 12-lipoxygenase Mm00545833_m1 Alox15 Arachidonate 15-lipoxygenase Mm01250458_m1 Axin2 Axin2 Mm00443610_m1 Mrps2 Mitochondrial ribosomal protein S2 (chob) Mm01962382_s91 Cx43 Gap junction membrane channel protein alpha 1 Mm01962382_s53 Dkk1 Dickkopf homolog 1 Mm00438422_m1 IL-1ß Interleukin-1β Mm00434228_m1 IL-6 Interleukin-6 Mm00446190_m1 Nkd2 Naked cuticle 2 homolog Mm00472240_m1 Sost Sclerostin Mm00470479_m1 Sfrp4 Secreted frizzled-related protein 4 Mm00840104_m1 TNF Tumor necrosis factor-α Mm00443258_m1 Tnfrsf11b Tumor necrosis factor receptor superfamily, member 11b (osteoprotogerin, OPG) Mm00435452_m1 Wisp1 WNT1 inducible signaling pathway protein 1 Mm00457574_m1 Wnt4 Wingless-related MMTV integration site 4 Mm00437341_m1 Wnt5a Wingless-related MMTV integration site 5A Mm00437347_m1 Wnt6 Wingless-related MMTV integration site 6 Mm00437353_m1 Wnt10b Wingless-related MMTV integration site 10B Mm00442104_m1 Wnt16 Wingless-related MMTV integration site 16 Mm00446420_m1 Open in a separate window TaqMan reagents used for quantification of mRNA by qPCR Statistics.

Techniques: Expressing

Journal: International Journal of Cancer

Article Title: The human PKP2 /plakophilin‐2 gene is induced by Wnt/β‐catenin in normal and colon cancer‐associated fibroblasts

doi: 10.1002/ijc.31104

Figure Lengend Snippet: Wnt3A regulates the expression of multiple genes in CCD‐18Co human colon myofibroblasts. ( a ) Validation using RT‐qPCR of a representative sample of six genes ( PTGER3 , ODZ3 , ILDR2, AMIGO2 , WNT16 and TRPA1 ) that were up‐ or downregulated by Wnt3A in RNA‐seq experiments. Cells were treated with either Wnt3A (+) or vehicle (–) for 24 hr. The results shown are means ± S.D. of three independent biological samples, each one determined in triplicate (*** p ≤ 0.001). ( b ) Wnt3A triggers the expression of a set of target genes encoding inhibitors of the Wnt/β‐catenin pathway. Cells were stimulated with either Wnt3A or vehicle for the indicated times and NKD1 , NKD2 , DKK2 , and APCDD1 expression was analysed by RT‐qPCR. Results are shown as fold over control at the indicated times and are means ± S.D. of three independent biological samples, each one determined in triplicate (n.s., non‐significant; *** p ≤ 0.001).

Article Snippet: We used TaqMan probes for PKP2 (Hs00428040_m1), PTGER3 (Hs00168755_m1), APCDD1 (Hs00537787_m1), DKK2 (Hs00205294_m1), NKD1 (Hs00263894_m1), NKD2 (Hs01108239_m1), WNT16 (Hs00365138_m1), TRPA1 (Hs00175798_m1), ODZ3 (Hs01111780_m1), ILDR2 (Hs01025498_m1), AMIGO2 (Hs00827141_g1), PKP1 (Hs00240873_m1), PKP3 (Hs00170887_m1), PKP4 (Hs00269305_m1), VIM (Hs00958111_m1) and ACTA2 (Hs00426835_g1) (Applied Biosystems).

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR, RNA Sequencing, Control

Journal: Oncology Reports

Article Title: Curcumin inhibits tumor epithelial-mesenchymal transition by downregulating the Wnt signaling pathway and upregulating NKD2 expression in colon cancer cells

doi: 10.3892/or.2016.4669

Figure Lengend Snippet: (A) Western blot analysis of the protein expression of naked cuticle homolog 2 (NKD2), β-catenin and TCF4 in SW620 cells after NKD2 siRNA transfection for 48 h. NC, negative control group; NKD2 siRNA 962 group; NKD2 siRNA 480 group. (B) The protein expression of NKD2, β-catenin and TCF4 in SW620 cells treated with the Wnt pathway activator (WAY 262611) for 48 h by western blot analysis. Con, control group; W (0.5 µ mol/l), WAY 262611 (0.5 µ mol/l) group; W (1 µ mol/l), WAY 262611 (1 µ mol/l) group.

Article Snippet: Curcumin was purchased from Sigma (Beijing, China), and NKD2 small-interfering RNA (siRNA) from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Control

Journal: Oncology Reports

Article Title: Curcumin inhibits tumor epithelial-mesenchymal transition by downregulating the Wnt signaling pathway and upregulating NKD2 expression in colon cancer cells

doi: 10.3892/or.2016.4669

Figure Lengend Snippet: (A) Western blot analysis of the protein expression of naked cuticle homolog 2 (NKD2), β-catenin and TCF4 in SW620 cells after NKD2 siRNA transfection for 48 h and curcumin treatment for 24 h. (B) Western blot analysis of the protein expression of NKD2, E-cadherin and vimentin in SW620 cells after curcumin treatment for 24 h and NKD2 siRNA transfection for 48 h. NC + c, negative control + curcumin group; NKD2 siRNA 962 + c, NKD2 siRNA 962 + curcumin group; NKD2 siRNA 480 + c, NKD2 siRNA 480 + curcumin group.

Article Snippet: Curcumin was purchased from Sigma (Beijing, China), and NKD2 small-interfering RNA (siRNA) from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Expressing, Transfection, Negative Control

Journal: Oncology Reports

Article Title: Curcumin inhibits tumor epithelial-mesenchymal transition by downregulating the Wnt signaling pathway and upregulating NKD2 expression in colon cancer cells

doi: 10.3892/or.2016.4669

Figure Lengend Snippet: (A) Western blot analysis of the protein expression of naked cuticle homolog 2 (NKD2) and chemokine receptor 4 (CXCR4) in SW620 cells after NKD2 siRNA transfection for 48 h and curcumin treatment for 24 h. * P<0.05, statistically significant. (B) RT-qPCR analysis of the mRNA expression of CXCR4 in SW620 cells after curcumin treatment for 24 h and NKD2 siRNA transfection for 48 h, * P<0.01 was considered statistically significant. NC + c, negative control + curcumin group; NKD2 siRNA 962 + c, NKD2 siRNA 962 + curcumin group; NKD2 siRNA 480 + c, NKD2 siRNA 480 + curcumin group.

Article Snippet: Curcumin was purchased from Sigma (Beijing, China), and NKD2 small-interfering RNA (siRNA) from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Negative Control

Journal: Traffic (Copenhagen, Denmark)

Article Title: Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

doi: 10.1111/tra.12642

Figure Lengend Snippet: PKA phosphorylates NKD2 at serine 223. A, MDCK cells stably expressing NKD2‐EGFP were labeled with 32 P‐ATP at 37°C for 2 hours, at which time cells were lysed and subjected to NKD2 immunoprecipitation (IP). The top panel (autoradiograph) shows incorporation of radiolabel in the NKD2 IP. The bottom panel shows NKD2 immunoblotting of the IPs as loading control from a parallel western blot. B, HEK293 cells stably expressing NKD2‐EGFP were incubated with 1 μM forskolin (FSK) (top panel) or 100 nM TPA (lower panel) for the indicated times. Cell lysates were subjected to NKD2 IP with the R44 antibody against NKD2 and then immunoblotted with pPKA or pPKC substrate motif antibodies, respectively. Membranes were later reprobed with NKD2 to confirm equal loading. As a positive control, AKAP12‐EGFP was expressed in the presence or absence of TPA. C, HEK293 cells transiently expressing NKD2‐EGFP or NKD1‐EGFP were incubated with 1 μM FSK for 5 minutes as indicated. Cell lysates were harvested and subjected to GFP IP, resolved on SDS‐PAGE, and probed with GFP, NKD1, NKD2 and pPKA substrate motif antibodies. D, Six serine residues predicted to be PKA phosphorylation sites within NKD2 were mutated individually to alanine (S > A). HEK293 cells transiently expressing EGFP‐tagged wild‐type (WT) NKD2 and indicated mutants were stimulated with 1 μM FSK for 5 minutes. Cell lysates were harvested and subjected to NKD2 IP, resolved on SDS‐PAGE and probed with antibodies against pPKA substrate motif antibody and NKD2. E, HEK293 cells transiently expressing NKD2‐EGFP and (S223A)NKD2‐EGFP were pretreated with the PKA inhibitor H89 (30 μM, 1 hour) and subsequently incubated with 1 μM FSK for 5 minutes as indicated. Cell lysates were subjected to IP and immunoblotted using the indicated antibodies. F, HEK293 cells transiently expressing NKD2‐EGFP were pretreated with H89 (30 μM, 1 hour) as indicated and then stimulated with VIP (100 nM), PGE 2 (100 ng/mL) or carbachol (CCh, 100 μM) for 5 minutes. Cell lysates were subjected to NKD2 IP and immunoblotted using the indicated antibodies. Corresponding total lysates were probed with β‐actin antibody as a loading control for (E and F)

Article Snippet: Anti‐human NKD2 antibody (R44) and TGFα polyclonal antibody were generated by Covance.

Techniques: Stable Transfection, Expressing, Labeling, Immunoprecipitation, Autoradiography, Western Blot, Control, Incubation, Positive Control, SDS Page, Phospho-proteomics

Journal: Traffic (Copenhagen, Denmark)

Article Title: Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

doi: 10.1111/tra.12642

Figure Lengend Snippet: FSK and VIP stimulate NKD2 S223 phosphorylation in polarized MDCK and Caco‐2 cells, respectively. A, MDCK cells stably expressing NKD2‐EGFP or (S223A)NKD2‐EGFP were grown on Transwell filters for 5 days. Cells were then either left untreated (CTL) or pretreated with H89 (30 μM, 1 hour) and/or stimulated with FSK (1 μM, 5 minutes) as indicated. Filters were paraformaldehyde‐fixed and immunostained for pS223‐NKD2 (red); NKD2‐EGFP fluorescence is shown in green. Confocal projections in the xz plane are shown. Scale bars: 10 μm. B, Parental Caco‐2 cells were grown on Transwell filters for 5 days. VIP (100 nM) was added to the basolateral compartment for 5 minutes (lower panel). Top panel shows untreated control (CTL). Filters were then paraformaldehyde‐fixed and stained for E‐cadherin (green), pS223‐NKD2 (red) and nuclei (DAPI, blue). Confocal projections in the xz plane are shown. Scale bars: 10 μm

Article Snippet: Anti‐human NKD2 antibody (R44) and TGFα polyclonal antibody were generated by Covance.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Fluorescence, Control, Staining

Journal: Traffic (Copenhagen, Denmark)

Article Title: Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

doi: 10.1111/tra.12642

Figure Lengend Snippet: NKD2 S223 phosphorylation‐deficient mutants reduce NKD2 stability and cell‐surface delivery of TGFα. A, MDCK cells stably expressing NKD2‐EGFP (upper panel) or (S223A)NKD2‐EGFP (lower panel) were treated with cycloheximide (CHX, 20 μg/mL) to block protein synthesis for the indicated times. Cell lysates were subjected to NKD2 and α‐tubulin immunoblotting. B, Results from three independent experiments in A were quantified and presented as relative intensity (RI) of NKD2 signal normalized to α‐tubulin signal. C, MDCK cells were transiently transfected with TGFα along with NKD2‐EGFP or (S223A)NKD2‐EGFP as indicated. These cells were pretreated with the PKA inhibitor H89 (30 μM) and/or the MMP inhibitor BB‐94 (5 μM) for 1 hour followed by FSK (1 μM, 5 minutes) as indicated at 37°C. Cells were then cooled and incubated with anti‐TGFα antibody under nonpermeabilized conditions at 4°C for 1 hour. Cells were then paraformaldehyde‐fixed and stained for p120. Fluorescence associated with TGFα, p120 and NKD2 is displayed in red, blue and green, respectively. Scale bars: 10 μm

Article Snippet: Anti‐human NKD2 antibody (R44) and TGFα polyclonal antibody were generated by Covance.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Blocking Assay, Western Blot, Transfection, Incubation, Staining, Fluorescence

Journal: Traffic (Copenhagen, Denmark)

Article Title: Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

doi: 10.1111/tra.12642

Figure Lengend Snippet: EGFR transactivation is dependent on PKA/NKD2/MMP‐mediated TGFα release. A, Conditioned media from overnight cultures (16‐24 hours) of MDCK cells transiently transfected with indicated plasmid constructs were collected. One hour prior to collection of conditioned media, the cultures were incubated with the antibodies indicated. Conditioned media were then added to serum‐starved A431 cells for 5 minutes. Recipient A431 cells were lysed and subjected to EGFR IP with subsequent immunoblotting for pY1173‐EGFR and total EGFR. B, Caco‐2 cells engineered to express TGFα under doxycycline control (Caco‐2 iTGFα) were incubated with the GPCR agonist VIP (100 nM) for the indicated times in the TGFα‐induced or ‐uninduced state and then lysed. Lysates were subjected to EGFR IP followed by immunoblotting for phospho‐tyrosine (pY) and EGFR. The image is representative of three independent experiments. C, Caco‐2 iTGFα cells in the induced state were preincubated with H89 (30 μM) or BB‐94 (5 μM) for 1 hour, stimulated with VIP (100 nM) and lysed after 1 minute as indicated. Lysates were subjected to EGFR IP followed by immunoblotting for pY and EGFR. Corresponding total lysates were immunoblotted for TGFα and β‐actin to show equal expression and loading

Article Snippet: Anti‐human NKD2 antibody (R44) and TGFα polyclonal antibody were generated by Covance.

Techniques: Transfection, Plasmid Preparation, Construct, Incubation, Western Blot, Control, Expressing

Journal: Traffic (Copenhagen, Denmark)

Article Title: Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

doi: 10.1111/tra.12642

Figure Lengend Snippet: AKAP12 and NKD2 interaction is necessary for NKD2 phosphorylation. A, Lysates from HEK293 cells transiently expressing NKD2 and AKAP12‐EGFP were prepared and subjected to IP with GFP or NKD2 antibodies followed by immunoblotting with AKAP12 and NKD2 antibodies as indicated. Right panel shows whole‐cell lysates immunoblotted for AKAP12, NKD2, and β‐actin as input controls. B, MDCK cells transiently expressing NKD2‐EGFP (green) and mCherry‐AKAP12 (red) were fixed and stained with DAPI (blue). Scale bar: 10 μm. C, Parental and CRISPR/Cas9‐based AKAP12 knockout (AKAP12 KO ) HEK293 cells were transiently transfected with NKD2‐EGFP and incubated in the presence or absence of FSK (1 μM) for 5 minutes. Cells were then lysed and subjected to immunoblotting with the indicated antibodies. D, Quantification of (C) from three independent experiments; * indicates P < 0.05

Article Snippet: Anti‐human NKD2 antibody (R44) and TGFα polyclonal antibody were generated by Covance.

Techniques: Phospho-proteomics, Expressing, Western Blot, Staining, CRISPR, Knock-Out, Transfection, Incubation

Journal: Traffic (Copenhagen, Denmark)

Article Title: Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

doi: 10.1111/tra.12642

Figure Lengend Snippet: AKAP12 and NKD2 interact intracellularly during GPCR‐induced NKD2 phosphorylation. A, Parental and AKAP12 KO HEK293 cells were incubated with FSK, VIP or PGE 2 for 30 seconds as indicated and were then fixed and stained for AKAP12 (red) and pS223‐NKD2 (green). Scale bars: 10 μm. B, FSK was added to HEK293 cells transiently expressing either wild‐type, (G2A)NKD2 or (S223A)NKD2. Lysates were immunoprecipitated with NKD2 or IgG control antibodies followed by immunoblotting for pS223‐NKD2 and NKD2. C, HEK293 cells transiently expressing NKD2‐EGFP were incubated with stapled inhibitory peptides (i‐peptide) RI‐STAD‐2 (RI‐selective) and STAD‐2 (RII‐selective) or their corresponding scrambled (scr) peptides (5 μM) for 1 hour and stimulated with FSK (0.5 μM) for 5 minutes. Cells were then lysed and subjected to IP with NKD2 antibodies followed by immunoblotting for pS223‐NKD2 and NKD2

Article Snippet: Anti‐human NKD2 antibody (R44) and TGFα polyclonal antibody were generated by Covance.

Techniques: Phospho-proteomics, Incubation, Staining, Expressing, Immunoprecipitation, Control, Western Blot