Journal: Cell death & disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression.

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: Fig. 3 B7-H3 promoted the expression of VEGFA in CRC. a The expression of angiogenesis-related genes was detected by RT-qPCR in shB7-H3 HCT116 and RKO cells. b Western blot analysis of B7-H3 and VEGFA in the sh-NC and shB7-H3 CRC cell lines. β-actin served as a loading control. c Representative images of IHC for VEGFA in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. d VEGFA protein expression based on the staining index of CRC specimens and matched normal tissues. e VEGFA protein expression is shown for patients stratified into B7-H3 low (median value) groups. f Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens (n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. NS no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After antigen retrieval with 10mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: Cell death & disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression.

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: Fig. 6 B7-H3 promoted angiogenesis via NF-κB/VEGFA pathway in Matrigel plugs in vivo. a, b CD31 (a) and VEGFA (b) protein expression based on their IHC staining index results in subcutaneous tumors formed by sh-NC-HCT116 and shB7-H3-HCT116 cells. N = 5. c, d CD31 (c) and VEGFA (d) protein expression based on their IHC staining index results in subcutaneous tumors formed by EV-HCT116 and B7-H3-HCT116 cells. N = 5. e, f CD31 (e) and VEGFA (f) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with BAY11–7082 (6 mg/kg). g, h CD31 (g) and VEGFA (h) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with bevacizumab (1 mg/kg). i, j CD31 (i) and VEGFA (j) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors co-treated with 3E8 (5 mg/kg) and BAY11–7082 (6 mg/kg) or bevacizumab (1 mg/kg). N = 5. The data represent the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After antigen retrieval with 10mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500).

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: Nature

Article Title: A growth-factor-activated lysosomal K + channel regulates Parkinson's pathology.

doi: 10.1038/s41586-021-03185-z

Figure Lengend Snippet: Fig. 1 | A growth-factor-activated lysosomal K+ channel, lysoKGF. a, Schematic of the recording of neuronal lysosomes. b, Currents (I) recorded at varying voltages (Ψ) from mouse hippocampal neurons with (starved) or without (fed) overnight starvation in DMEM containing no B27 nutrient. c, Currents from neurons with starvation followed by refeeding (in DMEM medium) with insulin (100 nM for 4 h), NGF (100 ng ml−1, 3 h) or BDNF (10 ng ml−1, 3 h). d, Reconstituting lysoKGF by TMEM175 transfection in HEK293T cells. Currents were recorded before, 2 h after starvation or after a 2-h starvation followed by refeeding with amino acids (10× for 10 min), with serum (10%, 4 h), with serum inactivated by boiling for 10 min or with insulin (100 nM, 4 h). e–i, Comparison between lysoKGF from wild-type and TMEM175-knockout neurons. e, Sequencing of the knock out, showing deletion of parts of exons 3

Article Snippet: TMEM175 (rabbit polyclonal, Proteintech, #19925-1-AP 1:1000), https://www.ptglab.com/products/TMEM175Antibody-19925-1-AP.htm, validated against knockout.

Techniques: Transfection, Comparison, Knock-Out, Sequencing

Journal: Nature

Article Title: A growth-factor-activated lysosomal K + channel regulates Parkinson's pathology.

doi: 10.1038/s41586-021-03185-z

Figure Lengend Snippet: Fig. 3 | AKT activates TMEM175 via catalysis-independent and interaction-dependent mechanisms. a, Association between native TMEM175 and AKT. Top two panels, total proteins prepared from wild-type and TMEM175-knockout mouse brains (lanes 1–3) or SH-SY5Y human neuroblastoma cells (lanes 4 and 5) were immunoprecipitated (IP) with anti-TMEM175 or with a control antibody (anti-UNC79) and blotted with anti-AKT and anti-TMEM175. Bottom two panels, total protein blotted for input control. b, Lysates from HEK293T cells transfected with GFP-tagged wild-type or mutant TMEM175 were immunoprecipitated with anti-GFP and blotted with anti-GFP or with anti-AKT. Whole-cell lysates (WCL) were also blotted for input control. c, Sequence alignment of the T338 region. The locations of T338 and K336 are indicated on the human TMEM175 structure (Protein Data Bank code (PDB) 6WCA). d, Pull down of AKT with human TMEM175– glutathione-S-transferase (GST) fusion protein. Cell lysates from human

Article Snippet: TMEM175 (rabbit polyclonal, Proteintech, #19925-1-AP 1:1000), https://www.ptglab.com/products/TMEM175Antibody-19925-1-AP.htm, validated against knockout.

Techniques: Knock-Out, Immunoprecipitation, Control, Transfection, Mutagenesis, Sequencing

Journal: Nature

Article Title: A growth-factor-activated lysosomal K + channel regulates Parkinson's pathology.

doi: 10.1038/s41586-021-03185-z

Figure Lengend Snippet: Fig. 5 | LysoKGF deficiency leads to accelerated spreading of pathogenic α-syn, loss of dopaminergic neurons and impaired motor function in mice. a, α-Syn spread assay. Cultured mouse hippocampal neurons were seeded with α-syn preformed fibrils and immunostained two weeks later with anti-pSer129 α-syn (pSyn, red), α-tubulin (green) and DAPI (blue). Bar graph shows pSyn signals normalized to the number of cells (n = 10 coverslips). Scale bar, 50 μm. AU, arbitrary units. b, Loss of dopaminergic neurons in TMEM175-knockout mice. Left, tyrosine hydroxylase (TH) immunostaining at the level of ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) from wild-type (top) and homozygous TMEM175-knockout (bottom) littermates (18–22 months

Article Snippet: TMEM175 (rabbit polyclonal, Proteintech, #19925-1-AP 1:1000), https://www.ptglab.com/products/TMEM175Antibody-19925-1-AP.htm, validated against knockout.

Techniques: Cell Culture, Knock-Out, Immunostaining

Journal: Nature communications

Article Title: Autophagy of the m 6 A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis.

doi: 10.1038/s41467-021-22469-6

Figure Lengend Snippet: Fig. 5 FTO regulates NEDD4L and arsenic tumorigenicity through m6A. a qPCR analysis of NEDD4L mRNA stability in As-T cells with or without FTO deletion and/or METTL14 knockdown (n = 3). b Immunoblot analysis of NEDD4L, METTL14, and FTO in cells as in a. c Tumor volume as in a following subcutaneous injection of cells as in a into NSG mice (n = 4). d Immunoblot analysis of NEDD4L and METTL3 in As-T cells with or without FTO knockout and/or METTL3 knockdown. e Soft agar assay of cells as in d (n = 3). f Immunoblot analysis of NEDD4L and METTL3 in cells with FTO deletion and METTL3 knockdown transfected with wild-type (WT) METTL3 or demethylase-inactive mutant METTL3 D395A. g qPCR analysis of NEDD4L mRNA level in cells as in f (n = 3). h Immunoblot analysis of NEDD4L, IGF2BP1, IGF2BP2, IGF2BP3, and FTO in As-T cells with or without FTO deletion in combination with transfection with siRNA targeting negative control (siNC) or all three IGF2BP1-3 (siALL). i qPCR analysis of NEDD4L mRNA level in cells as in h (n = 3). j qPCR analysis of NEDD4L mRNA stability in cells as in h (n = 3). k CLIP-qPCR showing the binding of IGF2BPs to the NEDD4L transcript in control and As-T cells (n = 3). All data were performed on n ≥3 biologically independent samples. Error bars are shown as mean ± S.D. (a, e, g, i–k) or mean ± S.E. c p-values of all data by two-tailed unpaired t-test are indicated.

Article Snippet: The cell lysate was mixed well, sonicated, and then incubated with 30 unit/ml of DNase I for 1 h. After centrifuging, the supernatant was used for IP experiments by using Magnetic beads (Protein A/G Magnetic Beads, 88802, Thermo ScientificTM), AntiFLAG® M2 Magnetic Beads (Sigma, M8823, 1:20), HA-Tag (C29F4) (Cell Signaling, #3724, 1:2000), DYKDDDDK Tag (9A3) (Cell Signaling, #8146, 1:2000), METTL3 (Proteintech, 15073-I-AP, 1:1000); Anti-p62/SQSTM1 (Sigma, P0067, 1:100), Anti-FTO (Santacruz, SC-271713, 1:200), Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) (Cell Signaling, #58802, 1:2000), and Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) (HRP Conjugate) (Cell signaling, #5127 HRP, 1:2000).

Techniques: Knockdown, Western Blot, Injection, Knock-Out, Soft Agar Assay, Transfection, Mutagenesis, Negative Control, Binding Assay, Control, Two Tailed Test