Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: High pY levels might affect the proliferation, migration and differentiation of lung fibroblasts. A , Changes in cell proliferation between hnLFs and hfLFs determined by CCK-8 kit (n = 7). *** P < 0.001. B-C , Assessing cell proliferation by EdU assay between hnLFs and hfLFs (n = 6). *** P < 0.001. D-G , Comparison of migration between hnLFs and hfLFs (n = 6). *** P < 0.001. H , Representative images of hnLFs and hfLFs under TEM. Arrows pointed to the lipid droplets in cytoplasm. I , Representative fluorescence images of Nile Red in hnLFs and hfLFs. J-K , The contractility of hnLFs and hfLFs in 3D collagen matrices (n = 6). *** P < 0.001. L , Phospho-RTK array analysis with 200 μg of lysates from hnLFs and hfLFs, which were serum-starved for 6 h. M , Quantification of phospho-RTK levels (average of two drops fold average of positive control). N-O , Western blot and quantification of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001. P-Q , Western blot and quantification of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001. R-S , Western blot and quantification of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Migration, CCK-8 Assay, EdU Assay, Comparison, Fluorescence, Positive Control, Western Blot

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder blocked multiple fibrosis associated pathways through interrupting pY-SH2 combination in pY mediated signal transmission. A-B , Phospho-RTK array analysis with 200 μg of lysates from hfLFs treated with 1 μM GST-SH2 WT or GST-SH2 TrM for 24 h. C , Western blot of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs after GST, GST-SH2 WT and GST-SH2 TrM incubation. D , Western blot of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. E , Western blot of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. F , hnLFs and hfLFs were incubated with 1 μM GST, GST-SH2 WT or GST-SH2 TrM for 24 h. The immunoprecipitation of EGFR and SHC was detected. G , Representative fluorescence images of EGFR and SHC colocalization in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM treatment. H , Immunoprecipitations of GRB2 with GAB1, SHC, EGFR and PDGFRβ. I , GST pull down assay showed the binding capacity of SH2 superbinder with pY in hnLFs and hfLFs. J , Representative fluorescence images of GST tag and pY colocalization in hnLFs and hfLFs after incubation with GST, GST-SH2 WT or GST-SH2 TrM. K-L , GST pull down assay showed the binding capacity of SH2 superbinder with RTKs (such as VEGFR2, PDGFRβ, EGFR and FGFR1) and adaptor proteins (such as GAB1, SHC and SRC) in hfLFs and hfLFs.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Transmission Assay, Western Blot, Incubation, Immunoprecipitation, Fluorescence, Pull Down Assay, Binding Assay

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: Diagrammatic representation of FGFR1 mutation from www.cbioportal.org .

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: FGFR1 protein expression by immunohistochemistry in SCLC and their correlations with prognosis. (A ) shows FGFR1 protein positive expression; ( B ) shows FGFR1 protein negative expression. Kaplan-Meier Survival analysis of FGFR1 protein-positive vs. FGFR1 protein-negative ( C and D ).

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: Clinicopathological data of the patients with SCLC and FGFR1 status

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Amplification

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: FGFR1 amplification by fluorescence in situ hybridization in SCLC and their correlations with prognosis. ( A ) shows FGFR1 amplification ( B ) shows FGFR1 non-amplification. Kaplan-Meier Survival analysis of FGFR1 amplified vs. non-amplified tumors ( C and D ).

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Amplification, Fluorescence, In Situ Hybridization

Journal: Cell Death & Disease

Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

doi: 10.1038/cddis.2017.322

Figure Lengend Snippet: OVOL1 regulated FLG expression in NHEKs. ( a and b ) Data are expressed as mean±S.E.M.; n =3 for each group; * P <0.05. ( a ) OVOL1 was overexpressed in NHEKs (OVOL1 OE cells) by electroporation of the plasmid containing an open reading frame of human OVOL1. FLG expression in the OVOL1 OE cells was analyzed by qRT-PCR. ( b ) OVOL1 was knocked down by transfection of OVOL1 siRNA into NHEKs (OVOL1 siRNA cells). FLG expression in OVOL1 siRNA NHEKs was analyzed by qRT-PCR. Mock-transfected NHEKs ( c ) and OVOL1 OE NHEKs ( d ) were stained with an anti-FLG antibody (primary antibody) and an Alexa Fluor 546-conjugated anti-mouse IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). Confocal laser scanning images revealed increased FLG expression (red) in OVOL1 OE NHEKs compared with mock-transfected NHEKs. ( e ) Isotype negative control. The scale bar is 25 μ m. The data are representative of experiments repeated three times with similar results

Article Snippet: The slices were then incubated with an anti-FLG antibody at room temperature for 1 h or an anti-OVOL1 antibody at 4 °C overnight, followed by incubation with a secondary antibody, N-Histofine Simple Stain AP (Nichirei Biosciences Inc.).

Techniques: Expressing, Electroporation, Plasmid Preparation, Quantitative RT-PCR, Transfection, Staining, Negative Control

Journal: Cell Death & Disease

Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

doi: 10.1038/cddis.2017.322

Figure Lengend Snippet: AHR activation induced by FICZ and Glyteer increased the expression of OVOL1 and FLG in a dose- and time-dependent manner in NHEKs. Data are expressed as mean±S.E.M.; n =3 for each group; * P <0.05. ( a , c , e , and g ) NHEKs were treated with FICZ or Glyteer at the indicated dose for 24 h. ( b , d , f , and h ) NHEKs were treated with FICZ (100 nM) or Glyteer (0.001%) for the indicated period. ( a – h ) Expression of FLG and OVOL1 was analyzed by qRT-PCR. ( i and j ) NHEKs were treated with FICZ or Glyteer at the indicated dose for 24 h or for the indicated period. Total cell lysates were prepared and subjected to western blot analysis with an anti-FLG antibody and anti-OVOL1 antibody. The data are representative of experiments repeated three times with similar results

Article Snippet: The slices were then incubated with an anti-FLG antibody at room temperature for 1 h or an anti-OVOL1 antibody at 4 °C overnight, followed by incubation with a secondary antibody, N-Histofine Simple Stain AP (Nichirei Biosciences Inc.).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

doi: 10.1038/cddis.2017.322

Figure Lengend Snippet: AHR regulated FLG expression via OVOL1 in NHEKs. ( a – d ) Data are expressed as mean±S.E.M.; n =3 for each group; * P <0.05. NHEKs were transfected with control siRNA or AHR siRNA and then treated with FICZ (100 nM) or Glyteer (0.001%) for 24 h. Expression of OVOL1 and FLG in the NHEKs was analyzed by qRT-PCR. ( e – k ) NHEKs transfected with control siRNA, AHR siRNA, or OVOL1 siRNA were treated with DMSO or FICZ (100 nM) for 24 h and then stained with an anti-FLG antibody (primary antibody) and an Alexa Fluor 546-conjugated anti-mouse IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). ( e ) Control siRNA-transfected NHEKs treated with DMSO, ( f ) control siRNA-transfected NHEKs treated with FICZ, ( g ) AHR siRNA-transfected NHEKs treated with DMSO, ( h ) AHR siRNA-transfected NHEKs treated with FICZ, ( i ) OVOL1 siRNA-transfected NHEKs treated with DMSO, and ( j ) OVOL1 siRNA-transfected NHEKs treated with FICZ. ( k ) Isotype negative control. Confocal laser scanning images revealed increased FLG expression (red) in control siRNA-transfected NHEKs treated with Glyteer ( f ) as compared with control siRNA-transfected NHEKs treated with DMSO ( e ); this upregulation was abrogated in AHR siRNA- or OVOL1 siRNA-transfected NHEKs treated with FICZ ( h and j ). The data are representative of experiments repeated three times with similar results. The scale bar is 25 μ m. ( l ) 3D-cultured NHEKs were treated with CH-223191 (10 μ M), FICZ (1 μ M), or CH-223191 (10 μ M) plus FICZ (1 μ M), or CH-223191 (10 μ M), Glyteer (0.01%), or CH-223191 (10 μ M) plus Glyteer (0.01%) for 48 h. Total cell lysates were prepared and subjected to western blot analysis with an anti-FLG antibody and an anti-OVOL1 antibody. The data are representative of experiments repeated three times with similar results

Article Snippet: The slices were then incubated with an anti-FLG antibody at room temperature for 1 h or an anti-OVOL1 antibody at 4 °C overnight, followed by incubation with a secondary antibody, N-Histofine Simple Stain AP (Nichirei Biosciences Inc.).

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

Journal: Cell Death & Disease

Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

doi: 10.1038/cddis.2017.322

Figure Lengend Snippet: Nuclear translocation of OVOL1 was likely to be inhibited in AD skin, leading to the reduced FLG expression in AD skin. Normal skin ( a ) and AD skin ( d ) were stained with hematoxylin and eosin. The scale bar is 100 μ m. Expression of FLG and OVOL1 in the epidermis of the same skin lesion was analyzed by IHC staining for FLG (red) or OVOL1 (red). The expression of FLG was observed in normal skin ( b ) and was low in AD skin ( e ). The expression of OVOL1 was observed mainly in the nuclei of keratinocytes in normal skin ( c ); however, nuclear OVOL1 expression was lower in AD skin ( f ). For semiquantitative analysis of IHC staining, microscopic visual fields of the samples from each group were randomly chosen and examined. In a high-power field (× 400 magnification), the nuclear-OVOL1-stained cells of the epidermis were counted, as were all the cells with hematoxylin staining. Nuclear OVOL1 expression was lower in AD skin (AD) compared with normal skin (NS) ( g ). NHEKs treated with DMSO ( h ), FICZ (100 nM) ( i ), IL-4 (10 ng/ml) ( j ), or FICZ plus IL-4 ( k) for 24 h were stained with an anti-OVOL1 antibody (primary antibody) and an Alexa Fluor 488-conjugated anti-rabbit IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). Confocal laser scanning images revealed that OVOL1 expression was noticeable mainly in the cytoplasm in a steady state ( h ) and that the AHR activation by FICZ induced nuclear translocation of OVOL1 ( i ). In contrast, IL-4 did not induce nuclear translocation of OVOL1, and the latter was retained in the cytoplasm ( j ). IL-4-mediated blockade of the nuclear translocation of OVOL1 was overridden by treatment with FICZ ( k ). ( l ) Isotype negative control. The scale bar is 25 μ m. The data are representative of experiments repeated three times with similar results. ( m ) NHEKs were treated with FICZ (100 nM) in the absence or presence of IL-4 (10 ng/ml) for 18 h. Cellular nuclear protein was extracted using a biochemical subcellular fractionation technique. The OVOL1 levels in the nuclear protein fraction of NHEKs were evaluated by western blotting. The activation of AHR by FICZ increased the nuclear OVOL1 expression; in contrast, IL-4 did not change nuclear expression of OVOL1. The IL-4-mediated blockade of the OVOL1 nuclear translocation was partially reversed by treatment with FICZ. The data are representative of experiments repeated three times with similar results

Article Snippet: The slices were then incubated with an anti-FLG antibody at room temperature for 1 h or an anti-OVOL1 antibody at 4 °C overnight, followed by incubation with a secondary antibody, N-Histofine Simple Stain AP (Nichirei Biosciences Inc.).

Techniques: Translocation Assay, Expressing, Staining, Immunohistochemistry, Activation Assay, Negative Control, Fractionation, Western Blot

Journal: Cell Death & Disease

Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

doi: 10.1038/cddis.2017.322

Figure Lengend Snippet: FICZ and Glyteer reversed the IL-4-induced decrease in FLG expression, which was dependent on OVOL1. Control siRNA- or OVOL1 siRNA-transfected NHEKs were treated with FICZ (100 nM) ( a ) or Glyteer (0.001%) ( b ) with or without IL-4 (10 ng/ml) for 24 h and then mRNA or total protein of the NHEKs were extracted. Expression of FLG in the NHEKs was analyzed by qRT-PCR. Data are expressed as mean±S.E.M.; n =3 for each group; * P <0.05. ( c ) Expression of FLG was analyzed by western blotting using the anti-FLG antibody. The data are representative of experiments repeated three times with similar results. The ability of FICZ or Glyteer to reverse the IL-4-induced downregulation of FLG was abrogated in OVOL1-knockdown NHEKs

Article Snippet: The slices were then incubated with an anti-FLG antibody at room temperature for 1 h or an anti-OVOL1 antibody at 4 °C overnight, followed by incubation with a secondary antibody, N-Histofine Simple Stain AP (Nichirei Biosciences Inc.).

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

Journal: Cell Death & Disease

Article Title: Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis

doi: 10.1038/cddis.2017.322

Figure Lengend Snippet: AHR activation induced by FICZ and Glyteer restores FLG expression via OVOL1 in the skin affected by atopic dermatitis (AD). 1. FICZ and Glyteer bind to AHR thereby leading to AHR activation. 2. Activated AHR induces upregulation and nuclear translocation of OVOL1. 3. The OVOL1 nuclear translocation of OVOL1 increases FLG expression. 4. IL-4 inhibits the nuclear translocation of OVOL1. 5. Inhibition of the nuclear translocation of OVOL1 downregulates FLG

Article Snippet: The slices were then incubated with an anti-FLG antibody at room temperature for 1 h or an anti-OVOL1 antibody at 4 °C overnight, followed by incubation with a secondary antibody, N-Histofine Simple Stain AP (Nichirei Biosciences Inc.).

Techniques: Activation Assay, Expressing, Translocation Assay, Inhibition