Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 1. NDP is expressed in a wide range of cancers, and is enriched in CNS tumors. (A and B) Analysis of NDP (A) and FZD4 (B) expression levels in primary human tumors from TCGA using the cBioportal web server. NDP expression was significantly enriched in GBM and lower-grade glioma relative to the average of all cancer types (upper graph), while FZD4 expression was comparable to other tumor types. ACC, adrenocortical carcinoma; AML, acute myeloid leukemia; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; PCPG, pheochromocytoma and paraganglioma; CS, carci- nosarcoma; ccRCC, clear cell renal cell carcinoma; chRCC, chromophobe renal cell carcinoma; pRCC, papillary renal cell carcinoma; VUS, variant of uncertain significance.

Article Snippet: For ectopic NDP and FZD4 overexpression experiments, we used TrueORF cDNA clones from Origene. pLenti-C-mGFP was used for NDP and pLenti-C-MYC-DDK was used for FZD4.

Techniques: Expressing, Variant Assay

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 2. NDP is expressed in GSCs and correlates with survival in neurological tumors. (A) Gene set enrichment analysis reveals correla- tion between NDP expression and “Glioblastoma Classical” and “Aging Brain” gene sets. (B) Kaplan-Meier analysis correlating NDP expression with patient survival in neurological cancers. (C) Expression of compo- nents of the NDP/FZD4 signaling axis in a panel of 9 patient-derived GSCs (left) and 3 primary fetal hNSC lines (right). Blue boxes, ASCL1lo GSC lines; red, ASCL1hi GSC lines indicate the GSC lines selected for functional analysis.

Article Snippet: For ectopic NDP and FZD4 overexpression experiments, we used TrueORF cDNA clones from Origene. pLenti-C-mGFP was used for NDP and pLenti-C-MYC-DDK was used for FZD4.

Techniques: Expressing, Derivative Assay, Functional Assay

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 4. Divergent effects of NDP and FZD4 knockdown on the proliferation indices of GSC cultures. (A and B) Representative images of ICC staining for Ki67 (red) and SOX2 (green) after NDP or FZD4 knockdown in ASCL1lo (G564) (A) and ASCL1hi (G472) (B) GSCs. Scale bars: 50 μm. (C and D) Quantification of the frequency of Ki67+ and SOX2+ cells after NDP and FZD4 knockdown in G564 (C) and G472 (D) GSCs. n = 3 (with the exception of Ki67 in G564; n = 9 because the cells are very large and the number of cells per field or vessel was quite small). Data are presented as mean ± SEM. *P < 0.05 by 1-way ANOVA.

Article Snippet: For ectopic NDP and FZD4 overexpression experiments, we used TrueORF cDNA clones from Origene. pLenti-C-mGFP was used for NDP and pLenti-C-MYC-DDK was used for FZD4.

Techniques: Knockdown, Staining

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 7. NDP knockdown inhibits Notch signaling in ASCL1hi GSCs. (A) Effects of recombinant Norrin treatment on the proliferation of G411 and G523 GSCs after 6 days in culture (n = 3). (B) Representative Western blot analysis for the indicated Notch pathway components in G411 and G523 GSCs after NDP knockdown. (C) Effects of Notch inhibition on the growth-promoting effects of NDP overexpression (OE) in G411 and or G523 GSCs after 6 days in cul- ture (n = 3). (D) Effect of NDP knockdown (KD) on the proliferation of G564 (ASCL1lo) GSCs after ASCL1 overexpression (n = 3). *P < 0.05 by Student’s t test (A) or 1-way ANOVA (C and D). Data are presented as mean ± SEM.

Article Snippet: For ectopic NDP and FZD4 overexpression experiments, we used TrueORF cDNA clones from Origene. pLenti-C-mGFP was used for NDP and pLenti-C-MYC-DDK was used for FZD4.

Techniques: Knockdown, Recombinant, Western Blot, Inhibition, Over Expression

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 9. Effects of NDP on progression of GBM in vivo depend on ASCL1 expression status. (A and B) Representative H&E staining of orthotopically xenografted G411 (ASCL1lo) (A) and G523 (ASCL1hi) GSC (B) tumors (dotted line, tumor outline). Shown are tile scans taken at ×2.5 magnification (left) and boxed regions are shown at higher magnification (×20) on the right. G411 GSCs form localized and G523 GSCs form diffuse tumors. Scale bars: 10 μm and 50 μm, respectively. (C and D) Kaplan-Meier survival analysis of mice orthotopically transplanted with G411 GSCs after NDP or FZD4 knockdown (C) and overexpression (D). (E and F) Kaplan-Meier survival analysis of mice orthotopically transplanted with G523 GSCs after NDP knockdown (E) and overexpres- sion (F). P < 0.05 by log-rank test (C–F).

Article Snippet: For ectopic NDP and FZD4 overexpression experiments, we used TrueORF cDNA clones from Origene. pLenti-C-mGFP was used for NDP and pLenti-C-MYC-DDK was used for FZD4.

Techniques: In Vivo, Expressing, Staining, Knockdown, Over Expression

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 10. NDP modulation results in selective advantage or disadvantage in xenografted tumors depending on ASCL1 expression status. (A and B) Representative IHC images of G411-derived tumors with NDP or FZD4 knockdown (A) or overexpression (B). Scale bars: 50 μm. (C and D) Representative IHC images of G523-derived tumors with NDP knockdown (C) or overexpression (D). Scale bars: 50 μm.

Article Snippet: For ectopic NDP and FZD4 overexpression experiments, we used TrueORF cDNA clones from Origene. pLenti-C-mGFP was used for NDP and pLenti-C-MYC-DDK was used for FZD4.

Techniques: Expressing, Derivative Assay, Knockdown, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: WNT5a induces fibroblast proliferation via WNT5a-FZD4/6-NFATc2 signaling. a, NFATc2 nuclear localization in fibroblasts induced by WNT5a. LL29 cells were serum-starved for 24 h and then stimulated with none (CON) or WNT5a (1 μg/ml) for 4 h. The cells were fixed for immunocytochemical analysis using anti-NFATc2 antibodies and Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Scale bar, 50 μm. b, inhibition of fibroblast proliferation by CsA. LL29 cells were treated with CsA (1 μg/ml) for 24 h and then stimulated with 1 μg/ml WNT5a for 12 h. Cell proliferation was determined by the BrdU assay. n = 3. c and d, effects of silencing FZD4, FZD6, and NFATc2 on fibroblast proliferation. LL29 cells were infected with a lentivirus expressing shRNA targeting FZD4, FZD6, or NFATc2 (siFZD4, siFZD6, or siNFACTc2, respectively) or a virus control (siCON) at an m.o.i. of 50 for 48 h. Protein levels were determined by Western blotting. The silenced cells were serum-starved for 24 h and then stimulated with 1 μg/ml WNT5a for 12 h. Cell proliferation was determined by the BrdU assay. n = 3–6. e and f, WNT5a and NFATc2 expression in IPF patients. Real-time PCR were performed using total RNA isolated from the lung tissues of IPF patients. n = 10 for the <50% FVC group, n = 10 for the 50–80% FVC group, and n = 8 for the >80% predicted. The expression level was relative to GAPDH. The results are presented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Staining, Inhibition, BrdU Staining, Infection, Expressing, shRNA, Virus, Control, Western Blot, Real-time Polymerase Chain Reaction, Isolation

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: miR-101 inhibits WNT5a-induced fibroblast proliferation. a, effect of overexpression of miR-101 on fibroblast proliferation. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h. The cells were serum-starved for 24 h and then stimulated for 12 h with 1 μg/ml WNT5a. Cell proliferation was determined by the BrdU assay (n = 5). b, histological staining of BrdU showing the effect of WNT5a and miR-101 on fibroblast proliferation. Scale bar, 50 μm. c, nuclear amount of NFATc2 was decreased by the overexpression of miR-101. LL29 cells were treated with a lentivirus expressing miR-101-1 and then stimulated with WNT5a (1 μg/ml). Nuclei were extracted using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Waltham, MA) to determine the expression of the NFATc2 and H1 proteins by Western blotting. d, overexpression of miR-101-1 suppressed WNT5a-induced NFATc2 mRNA expression in LL29 cells by real-time PCR analysis. The expression levels were relative to GAPDH. n = 3. e, NFATc2 nuclear localization in the absence of FZD4 and FZD6. LL29 cells were split into a chamber slide at 2,000 cells per chamber. After a 24-h culture, cells were infected with lentiviral shFZD4 and shFZD6 (m.o.i. 25 for each) or shCon at m.o.i. = 50 for 48 h. Cells were serum-starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 4 h. The cells were fixed for immunocytochemical analysis using anti-NFATc2 antibodies and Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Scale bar, 50 μm. The results are represented as the mean ± S.E. ANOVA followed by Tukey's HSD test was performed for multiple comparisons. , p < 0.05; **, p < 0.01.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression, Expressing, Virus, Control, BrdU Staining, Staining, Extraction, Western Blot, Real-time Polymerase Chain Reaction, Infection

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Identification of FZD4, FZD6, and TGFBR1 as miR-101 targets. a, 3′-UTR reporter assay: FZD4-UTR-S1 (position 1–1,432) containing miR-101-binding site 1 (position 537–543), FZD4-UTR-S2 (position 4,178–5,463) containing miR-101-binding site 2 (position 1,240–1,246), full length of FZD6-UTR, TGFBR1-UTR-S1 (position 66–1,524) containing miR-101-binding site 1 (position 460–466), or TGFBR1-UTR-S2 (position 3,210–4,749) containing miR-101-binding site 2 (position 3,993–3,999) were co-transfected into HEK 293T cells with miR-101-1. The relative luciferase activities were measured using the Dual-Luciferase® reporter assay system. n = 3. b and c, effect of overexpressing or knockdown miR-101 on the protein expression of FZD4, FZD6, and TGFBR1 in fibroblasts. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h (b) or CCD-8Lu fibroblasts were treated with a lentivirus expressing anti-miR-101 or the virus control (Anti-CON) at an m.o.i. of 50 for 72 h. c, Western blotting was performed to determine the protein expression of FZD4, FZD6, and TGFBR1. The protein samples used here were the same as mentioned in Fig. 7c. The GAPDH immunoblot shown in Fig. 7c was used again here. d, overexpression of miR-101 inhibits FZD4, FZD6, and TGFBR1 mRNA expression in fibroblasts. LL29 cells were treated with lentiviral miR-101 or the virus control at an m.o.i. of 50 for 48 h. The mRNA expression of FZD4, FZD6, and TGFBR1 was determined by real-time PCR and normalized to GAPDH. The results are presented as the mean ± S.E. n = 3. e, real-time PCR showing the increased mRNA levels of FZD4, FZD6, and TGFBRR1 by anti-miR-101. CCD-8Lu fibroblasts were treated with lentiviral anti-miR-101 or the virus control at an m.o.i. of 50 for 72 h. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. f–h, mRNA expression of FZD4, FZD6, and TGFBR1 in IPF patient lungs. The mRNA levels of FZD4, FZD6, and TGFBR1 were determined by real-time PCR and normalized to β-actin. n = 8–10. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Reporter Assay, Binding Assay, Transfection, Luciferase, Knockdown, Expressing, Virus, Control, Western Blot, Over Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Overexpression of FZD4, FZD6, or TGFBR1 rescues miR-101-mediated inhibition of fibroblast proliferation and activation. a–d, knockdown of TGFBR1 repressed the TGF-β1-induced mRNA expression of α-SMA, COL1A1, COL3A1, and COL4A1 in fibroblasts. LL29 cells were treated with lentivirus shRNA-TGFBR1 or the virus control at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. The α-SMA, COL1A1, COL3A1, and COL4A1 mRNA expression levels were determined by real-time PCR. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. The results are presented as the mean ± S.D. **, p < 0.01. e and f, overexpression of FZD4 or FZD6 rescues the miR-101-mediated inhibition of fibroblast proliferation. LL29 cells were split into 96-well plates at 3,000 cells per well. After a 24-h culture, cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con miR-101, GFP, FZD4, and FZD6 at m.o.i. = 25 each, virus control (VC) = miR-Con + GFP) for 48 h. Cells were starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 12 h. FZD4 and FZD6 expression was determined by Western blotting by using anti-GFP antibody. Cell proliferation was determined by BrdU assay. Data were presented as mean ± S.E. Statistical analysis was performed by ANOVA and followed by Tukey's HSD test. n = 4. **, p < 0.01. g–j, overexpression of TGFBR1 rescues miR-101-mediated inhibition of α-SMA, COL1A1, and COL3A1 mRNA expression. LL29 cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con, miR-101, GFP, and TGFBR1 at m.o.i. = 25 each, virus control = miR-Con + GFP) for 48 h. Cells were then stimulated with TGFβ1 (5 ng/ml) for 48 h. Cells were collected for real-time PCR. The results are presented as the mean ± S.E., n = 3. ANOVA followed by Tukey's HSD test was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression, Inhibition, Activation Assay, Knockdown, Expressing, shRNA, Virus, Control, Real-time Polymerase Chain Reaction, Infection, Western Blot, BrdU Staining

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Adenovirus-mediated miR-101 gene transfer attenuates bleomycin-induced pulmonary fibrosis. On day 1, an adenovirus expressing miR-101-1 or the control adenovirus (VC) (5 × 109 pfu per mouse) was delivered into the lungs of 6–8-week-old C57BL/6 mice by nasal instillation. On day 2, bleomycin (Bleo) (0.06 units per mouse) or the same volume of saline (Sal) was intranasally instilled into the lungs. On day 15, the mice were subjected to the analysis of respiratory mechanics by Flexivent and then sacrificed. The left lungs were collected for RNA and protein analysis, and the right lungs were fixed for histological analysis. a, real-time PCR analysis showing adenovirus-mediated overexpression of miR-101 in the mouse lung. The expression levels were relative to U6. b, H&E staining showing the fibrotic changes in the mouse lung induced by bleomycin. miR-101 attenuated the fibrotic changes in bleomycin-treated mouse lungs. Scale bar, 100 μm. c, Ashcroft score grade of pulmonary fibrosis. d, lung collagen content as determined using the QuickZyme hydroxyproline assay kit. e and f, real-time PCR analysis showing the increased COL1A1 and COL3A1 mRNA levels in bleomycin-treated mouse lungs and miR-101 suppression of the increase of COL1A1 and COL3A1 mRNA levels. The expression levels were relative to GAPDH. g and h, analysis of lung mechanics by Flexivent analysis. Elastance (Ers) was measured in a single-compartment model. h was measured in the Constant-Phase model. i and j, NFATc2, FZD4, FZD6, and TGFBR1 mRNA expression was increased in bleomycin-treated mouse lungs, and the increase was suppressed by miR-101 treatment as determined by real-time PCR analysis. The expression level was relative to GAPDH. n = 5 for saline + VC and saline + miR-101; n = 7–10 for bleomycin + VC and bleomycin + miR-101. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Expressing, Control, Saline, Real-time Polymerase Chain Reaction, Over Expression, Staining, Hydroxyproline Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Primers for the construction of plasmids FW is forward, and RE is reverse.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate receptor 2 inhibition ameliorates familial exudative vitreoretinopathy models

doi: 10.1016/j.jbc.2025.111107

Figure Lengend Snippet: FZD4 inhibition prevents primary human retinal microvascular endothelial cell (hRMEC) tubularization. A , Microscopy of tubularization in primary hRMECs treated with FZD4 shRNA. B , computational analysis of vascularization in both FZD4 shRNA treated and control. Eight different parameters were examined and measured using FIJI ( , ). C , microscopy of tubularization in hRMECs due to expression of the patient derived dominant-negative FZD4 Met493-Trp49del allele. D , computational analysis of vascularization in both overexpression of the FZD4 variant and the control. There is significant reduction in all eight examined parameters when the FZD4 variant is overexpressed. Ordinary one-way ANOVAs with multiple comparisons were performed on the data. ∗ p < 0.05, ∗∗ p < 0.01; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Size bar is 500 μM.

Article Snippet: Human shRNA Plasmid Kit (Origene, catalog number TL312883) was used for shRNA knockdown of FZD4 expression.

Techniques: Inhibition, Microscopy, shRNA, Control, Expressing, Derivative Assay, Dominant Negative Mutation, Over Expression, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate receptor 2 inhibition ameliorates familial exudative vitreoretinopathy models

doi: 10.1016/j.jbc.2025.111107

Figure Lengend Snippet: The S1PR inhibitor JTE-013 rescues FZD4 mediated primary human retinal microvascular endothelial cell (hRMEC) tubularization defects. A , microscopy of vascular formation in the primary hRMECs treated with FZD4 shRNA and the control (shRNA scramble) with and without JTE-013. Cells were seeded onto a basement membrane and incubated for 6 h at 37 °C prior to imaging. JTE-013 was added at the time of seeding. B , parameters measured by the angiogenesis plugin on FIJI and their statistical analysis. Ordinary one-way ANOVAs with multiple comparisons were performed on the data. ∗ p < 0.05, ∗∗ p < 0.01; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Size bar is 500 μM.

Article Snippet: Human shRNA Plasmid Kit (Origene, catalog number TL312883) was used for shRNA knockdown of FZD4 expression.

Techniques: Microscopy, shRNA, Control, Membrane, Incubation, Imaging

Journal: The FASEB Journal

Article Title: Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families

doi: 10.1096/fj.201700144R

Figure Lengend Snippet: Primers used for indel-nested PCR 1 for validation of mutations generated by sgRNAs

Article Snippet: FZD4 , pCMV6-XL6 FZD4 , Origene , SC115479 , .

Techniques: Biomarker Discovery, Generated

Journal: The FASEB Journal

Article Title: Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families

doi: 10.1096/fj.201700144R

Figure Lengend Snippet: Constructs used in this study

Article Snippet: FZD4 , pCMV6-XL6 FZD4 , Origene , SC115479 , .

Techniques: Construct, Luciferase

Journal: The FASEB Journal

Article Title: Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families

doi: 10.1096/fj.201700144R

Figure Lengend Snippet: TaqMan qPCR primers and probes

Article Snippet: FZD4 , pCMV6-XL6 FZD4 , Origene , SC115479 , .

Techniques:

Journal: The FASEB Journal

Article Title: Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families

doi: 10.1096/fj.201700144R

Figure Lengend Snippet: Multiple targeting of up to 5 FZD homologous genes with single sgRNA for FZD1,2,4,5,7,8 sgRNA

Article Snippet: FZD4 , pCMV6-XL6 FZD4 , Origene , SC115479 , .

Techniques:

Journal: Clinical Cancer Research

Article Title: ZIP4 Regulates Pancreatic Cancer Cell Growth by Activating IL-6/STAT3 Pathway through Zinc Finger Transcription Factor CREB

doi: 10.1158/1078-0432.ccr-09-2405

Figure Lengend Snippet: Fig. 2. Phosphorylation of STAT3 in MIA-ZIP4 and ASPC-shZIP4 cells. Stable MIA-V, MIA-ZIP4, ASPC-shV, ASPC-shZIP4, BxPC-shV, and BxPC-shZIP4 cells were examined for the expression and phosphorylation of STAT3 protein. Briefly, cell lysates were extracted and probed with anti-p(tyr)STAT3 (1:1,000) and anti-STAT3 (1:1,000) antibodies.

Article Snippet: ZIP4 overexpressing and shRNA-silenced stable cells were selected in MIA PaCa-2, ASPC-1, and BxPC-3 cells with retrovirus vectors (Origene) as previously described (5, 6).

Techniques: Phospho-proteomics, Expressing

Journal: Clinical Cancer Research

Article Title: ZIP4 Regulates Pancreatic Cancer Cell Growth by Activating IL-6/STAT3 Pathway through Zinc Finger Transcription Factor CREB

doi: 10.1158/1078-0432.ccr-09-2405

Figure Lengend Snippet: Fig. 5. ZIP4 upregulates the IL-6 promoter activity through phosphorylating CREB in pancreatic cancer cells. A, schematic diagram of IL-6 promoter luciferase reporter construct. B, IL-6 promoter activity. Wild-type IL-6 promoter reporter was cotransfected with ZIP4 or vector control plasmid into 293 cells, the IL-6 promoter activity was determined by a chemiluminescence reader. C, phosphorylation of CREB in MIA-ZIP4 cells. Stable MIA-V and MIA-ZIP4 cells were examined for the expression and phosphorylation of CREB. Briefly, cell lysates were extracted and probed with anti-pCREB (1:1,000) and anti-CREB (1:1,000) antibodies.

Article Snippet: ZIP4 overexpressing and shRNA-silenced stable cells were selected in MIA PaCa-2, ASPC-1, and BxPC-3 cells with retrovirus vectors (Origene) as previously described (5, 6).

Techniques: Activity Assay, Luciferase, Construct, Plasmid Preparation, Control, Phospho-proteomics, Expressing