Journal: Scientific Reports

Article Title: YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway

doi: 10.1038/s41598-024-61157-5

Figure Lengend Snippet: Overexpression of YANK2 promotes cell growth and tumorigenicity of glioma. ( A ) Expression of YANK2 were shown in U87MG-shYANK2 and Hs683-shYANK2 cells by WB (All bands are the result of the same sample, Fig. 2A is the cropped picture, and the original picture is shown in Supplementary raw data Figure A); ( B ) Cell clone formation assays were performed in U87MG- or Hs683-shYANK2 cells with representative images (left for plate clone; middle for soft agar) and quantification of spheres with a volume greater than 2 μm in soft agar (right) was shown. The data are presented as the mean ± SEM of three replications. * P < 0.05; ** P < 0.01; *** P < 0.001; ( C ) Proliferation of H4- or U118MG-YANK2 stable cells were detected by MTT assay. Insert showed verification of the cell lines identified by WB (All PVDF bands are transferred and tested in the same sample); ( D ) Colony formation assay in H4- or U118MG-YANK2 cells with images acquired after 14 days, n = 3; ( E ) The representative bioluminescence images of BALB/c nude mice with tumors derived from U87MG-LUC-shYANK2 on day 14 (left upper). Left lower showed verification of the cell lines identified by WB. Colored scale bar represents photons/s/cm /steradian. Kaplan–Meier survival curves of mice are shown (right). 2 × 10 5 cells per mouse, n > 6. (Fig. 2E is the cropped picture, and the original picture is shown in supplemental raw data Figure E); ( F ) Hematoxylin and eosin stained (HE) coronal brain sections of tumor xenograft (upper) and the quantified results (lower), IHC of Ki-67 in tumor slices (middle). Scale bars for HE: 2.5 mm. Scale bars for IHC: 100 μm, images of representative tumors were shown. (All PVDF bands are transferred and tested in the same Wb experiment); ( G ) Representative pictures of cell clone formation assays which were performed in GL261-LUC-YANK2 cells and control groups (upper for verification of the cell lines identified by WB; middle for plate clone; lower for soft agar). (All PVDF bands are transferred and tested in the same Wb experiment); ( H ) The representative bioluminescence images of C57 BL/6J mice with tumors derived from GL261-LUC-YANK2 on day 14 (left). Colored scale bar represents photons/s/cm /steradian. Kaplan–Meier survival curves of mice were shown (right). 2 × 10 5 cells per mouse, n > 6; in ( A – H ), Mock and pHAGE were vector control. All bar plot data are means ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The five human glioma cell lines A172, H4, U118MG, U87MG and U373, the mouse glioma cell line GL261, the rat glioma cell line C6 and the human embryonic renal cell line HEK293T were purchased on ATCC.

Techniques: Over Expression, Expressing, MTT Assay, Colony Assay, Derivative Assay, Staining, Control, Plasmid Preparation

Journal: Scientific Reports

Article Title: YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway

doi: 10.1038/s41598-024-61157-5

Figure Lengend Snippet: Fyn directly binds with and phosphorylates YANK2 in vitro and ex vivo . ( A ) Potential phosphorylated tyrosine sites and upstream kinase of YANK2 were predicted by prediction of PK-specific phosphorylation site website; ( B ) Pearson correlation analysis mRNA levels between Fyn and YANK2 in glioma with IDH-WT (data comes from CGGA RNA-sequencing dataset). Pearson correlation test, R and P values are presented; ( C , D ) The level of YANK2 in Hs683 and U87MG threated with different concentration PP2 (Fyn inhibitor) for 24 h was detected by WB ( C ) and the densities of YANK2/Tubulin in Hs683 and U87MG were determined by densitometry ( D ). (All bands are the result of the same sample); ( E ) Expression of YANK2 and Fyn were shown in U118MG- or U87MG-sgFyn cells by WB. (All bands are the result of the same sample); ( F ) pBind-YANK2 or pHAGE-Myc-Fyn-Flag plasmid was transfected or co-transfected into HEK293T cells for 48 h, immunoprecipitated with an anti-Flag, and then probed with anti-YANK2 antibody. (All bands are the result of the same sample); ( G ) pCMV-His-YANK2-Flag or pHAGE-Myc-Fyn-Flag plasmid was transfected or co-transfected into HEK293T for 48 h, pulldown with Ni-NTA beads and then probed with anti-Myc antibody. (All bands are the result of the same sample); ( H ) Endogenous YANK2 was immunoprecipitated from U87MG cells and then probed with anti-Fyn antibody. (All bands are the result of the same sample); ( I ) pCMV-His-YANK2-Flag and pHAGE-Myc-Fyn-Flag (WT, K299M, Y531F) plasmid was co-transfected into HEK293T for 48 h, pulldown with Ni-NTA beads and then probed with anti-p-Tyr antibody. (All bands are the result of the same sample); ( J ) Fyn phosphorylates YANK2 in vitro. Active Fyn was obtained by IP with anti-Flag from HEK293T cells which were transiently transfected with pHAGE-Myc-Fyn-Flag for 48 h and stimulated with EGF (80 ng/ml, 30 min), and then a kinase assay was performed with His-YANK2-His purified from bacteria as substrate, and phosphorylation of YANK2 was detected using anti-p-Tyr antibody by WB. (All bands are the result of the same sample). All bar plot data are means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The five human glioma cell lines A172, H4, U118MG, U87MG and U373, the mouse glioma cell line GL261, the rat glioma cell line C6 and the human embryonic renal cell line HEK293T were purchased on ATCC.

Techniques: In Vitro, Ex Vivo, Phospho-proteomics, RNA Sequencing, Concentration Assay, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Kinase Assay, Purification, Bacteria

Journal: Scientific Reports

Article Title: YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway

doi: 10.1038/s41598-024-61157-5

Figure Lengend Snippet: YANK2 phosphorylation by Fyn at Y110 promotes glioma growth by increasing its stability. ( A ) Mass spectrometry (MS) assay was carried out to analyze the phosphorylation modification of YANK2 after an in vitro kinase assay in which Fyn served as an active kinase and His-YANK2-His protein as substrate. The annotated MS/MS spectrum of the phospho-peptide containing the Tyr110 phosphorylated residue of YANK2 was shown. (All bands are the result of the same sample); ( B ) Active Fyn was obtained by IP with anti-Flag from HEK293T cells which were transiently transfected with pHAGE-Myc-Fyn-Flag for 48 h and stimulated with EGF (80 ng/ml, 30 min), and then a kinase assay was performed with His-YANK2-His (WT, Y110F) purified from bacteria as substrate, and phosphorylation of YANK2 was detected using anti-p-Tyr antibody by WB. (All bands are the result of the same sample, Fig. 4B is the cropped picture, and the original picture is shown in supplementary raw data Figure B); ( C ) pCMV-His-YANK2-Flag (WT, Y110F) or pHAGE-Myc-Fyn-Flag plasmid was transfected or co-transfected into HEK293T for 48 h, pulldown with Ni-NTA beads and then probed with anti-p-Tyr antibody. (All bands are the result of the same sample, Fig. 4C is the cropped picture, and the original picture is shown in supplemental raw data Figure C); ( D ) Amino acid sequence of Y110 residue (red) in YANK2 is highly conserved in different species; ( E , F ) The time-dependent stability of YANK2 in H4-YANK2-Flag (WT, Y110D, Y110F) stable cells treated with CHX (100 μg/ml) was analyzed with anti-Flag by WB ( E ) and the densities of flag/tubulin were determined by densitometry ( F ). (All bands are the result of the same sample); ( G ) Plate clone formation assays were performed in U373- or H4-YANK2 (WT, Y110F) stable cells with representative images; ( H , I ) Clone formation assays by soft agar were performed in U373- or U118MG-YANK2 (WT, Y110F) stable cells with representative images ( H ) and quantification of spheres with a volume greater than 2 μm in soft agar was shown ( I ). The data are presented as the mean ± SEM of three replications. * P < 0.05; ** P < 0.01; ( J , K ) The representative bioluminescence images of C57 BL/6J mice with tumors derived from GL261-LUC-YANK2 (WT, Y110F) on day 14 ( J ). Colored scale bar represents photons/s/cm 2 /steradian. Kaplan–Meier survival curves of mice are shown ( K ). 2 × 10 5 cells per mouse, n > 6; in ( J , K ), pHAGE was vector control. All WB analyses are presented as mean ± SEM and representative pictures of three independent experiments. * P < 0.05; ** P < 0.01.

Article Snippet: The five human glioma cell lines A172, H4, U118MG, U87MG and U373, the mouse glioma cell line GL261, the rat glioma cell line C6 and the human embryonic renal cell line HEK293T were purchased on ATCC.

Techniques: Phospho-proteomics, Mass Spectrometry, Modification, In Vitro, Kinase Assay, Tandem Mass Spectroscopy, Residue, Transfection, Purification, Bacteria, Plasmid Preparation, Sequencing, Derivative Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-4 receptor signaling modulates neuronal network activity

doi: 10.1084/jem.20211887

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: , CD31 , CD31-Pe-Vio770 , ms , Miltenyi , 130-119-894.

Techniques: Marker, Western Blot, Tandem Mass Spectroscopy

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-4 receptor signaling modulates neuronal network activity

doi: 10.1084/jem.20211887

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: Oligodendrocyte marker O4 , O4-APC , ms , Miltenyi , 130-119-155.

Techniques: Marker, Western Blot, Tandem Mass Spectroscopy