Journal: Stem Cell Research & Therapy

Article Title: The effect of mitochondrial fusion on chondrogenic differentiation of cartilage progenitor/stem cells via Notch2 signal pathway

doi: 10.1186/s13287-022-02758-7

Figure Lengend Snippet: Schematic illustration of the proposed mechanisms for chondrogenic differentiation of CPSCs and the interaction of mitochondrial fusion with NOTCH2 signaling pathway

Article Snippet: Blotting was performed using primary antibodies: Col2 (1:1000)(rabbit; no. ab34712; Abcam), GAPDH (1:1000) (rabbit; no. ab70699; Abcam), SOX-9 (1:1000) (rabbit; no. ab185966Ab; Abcam), Acan (1:1000) (rabbit; no. ab36861; Abcam), MFN2 (1:200) (mouse; no. Sc-100560; Santa Cruz) NICD (1:500) (rabbit; no. 10062-2-AP; Proteintech; Wuhan, China), MFN1(1:500) (rabbit; no. 13798-1-AP; Proteintech), NOTCH2 (1:1000) (rabbit; no. 5737; Cell signaling Tec.), HES1(1:1000) (rabbit; no. 11988; Cell signaling Tec), β-actin (1:200) (mouse; no. Sc-8432; Santa Cruz).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: The effect of mitochondrial fusion on chondrogenic differentiation of cartilage progenitor/stem cells via Notch2 signal pathway

doi: 10.1186/s13287-022-02758-7

Figure Lengend Snippet: Mfn2 - OE suppressed NOTCH2 signaling pathway. A Protein level expression of Notch2, smad3, smad4, and β -catenin on day 0, 3, 7 and 14 of chondrogenesis. B Relative quantitative analysis of the proteins level expression. C , D Comparison of the effects of relevant expression of Notch2, smad3, smad4, and β-catenin signaling pathways on undifferentiated CPSCs using western blot between OE-NC, OE, KD-NC, and KD group, and relative quantitative analysis. E , F Comparison of the effects of relevant expression of Notch2, smad3, smad4, and β-catenin signaling pathways on differentiated CPSCs using western blot between OE-NC, OE, KD-NC, and KD group, and relative quantitative analysis. The data are expressed as mean ± standard deviation, N = 3. *P less than 0.05 versus OE-NC, KD or KD-NC. OE over-expression of Mfn2 , OE-NC negative control group of Mfn2 over-expression, KD knockdown of Mfn2 , KD-NC negative control of Mfn2 knockdown

Article Snippet: Blotting was performed using primary antibodies: Col2 (1:1000)(rabbit; no. ab34712; Abcam), GAPDH (1:1000) (rabbit; no. ab70699; Abcam), SOX-9 (1:1000) (rabbit; no. ab185966Ab; Abcam), Acan (1:1000) (rabbit; no. ab36861; Abcam), MFN2 (1:200) (mouse; no. Sc-100560; Santa Cruz) NICD (1:500) (rabbit; no. 10062-2-AP; Proteintech; Wuhan, China), MFN1(1:500) (rabbit; no. 13798-1-AP; Proteintech), NOTCH2 (1:1000) (rabbit; no. 5737; Cell signaling Tec.), HES1(1:1000) (rabbit; no. 11988; Cell signaling Tec), β-actin (1:200) (mouse; no. Sc-8432; Santa Cruz).

Techniques: Expressing, Comparison, Protein-Protein interactions, Western Blot, Standard Deviation, Over Expression, Negative Control, Knockdown

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Correlation between NOTCH1–4 expression and Gleason score or prognosis in PCa patients. (A) UALCAN database analysis showing that the transcriptional level of NOTCH3 and NOTCH4 is significantly enhanced with increasing Gleason score. (B) The Mantel–Cox test with GEPIA2 showing that the expression of NOTCH3 and NOTCH4 exhibited a positive correlation with prognosis in PCa patients. (C) Cox regression analysis showing that patients in the low NOTCH1, 3 and 4 groups had a significant better disease-free survival (DFS) than those in the high group (P=4.8e-2, 5.6e-3, and 2.0e-2, respectively). (D) Cox regression analysis showing that there was no significant difference in overall survival (OS) between high and low NOTCH family genes’ expression. *, P<0.05, **, P<0.01, ***, P<0.001 and ****, P<0.0001. GS, Gleason score; PRAD, prostate adenocarcinoma; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Characterization of genetic alterations and mutations of NOTCH1–4. (A) Mutational frequencies of NOTCH1, NOTCH2, NOTCH3 and NOTCH4 in PCa were 7%, 8%, 5%, and 5%, respectively. (B) Top 10 most significant mutation-related genes of NOTCH1–4. (C) Wayne chart showing the shared mutation-related genes of NOTCH1–4. (D) Bar graph showing the top 20 results from the GO enrichment analysis. (E) GO enrichment analysis showing the gene networks. GO, gene ontology; PCa, prostate cancer.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Mutagenesis

Journal: Translational Andrology and Urology

Article Title: Evaluation of NOTCH family genes’ expression and prognostic value in prostate cancer

doi: 10.21037/tau-22-281

Figure Lengend Snippet: Correlation between differently expressed NOTCH1–4 and lymphatic metastasis or immune cell infiltration. (A) TIMER database analysis showing that patients with N1 stage had significant higher expression of NOTCH3 than those with N0 stage (P<0.05, N0 = no lymphatic metastasis, N1 = lymphatic metastasis). (B) Associations between NOTCH1–4 and lymphocytes infiltration in PCa patients. *, P<0.05, ***, P<0.001 and ****, P<0.0001. ns, no significant. PCa, prostate cancer; PRAD, prostate adenocarcinoma; TIMER, tumor immune estimation resource.

Article Snippet: Antibodies against NOTCH1 (Proteintech #20687-1-AP), NOTCH2 (Proteintech #28580-1-AP), NOTCH3 (Proteintech #55114-1-AP), and NOTCH4 (Abclonal #A8303) were incubated overnight at 4 °C.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Notch was found up-regulated after azelaic acid (AZA) treatment. (A) 120 differential expressed proteins associated in 52 functions after AZA treatment by protein mass spectrometry analysis in the heat map. (B) GO annotation with 528 differential expressed proteins. (C, D) THP-1, Molm-13, NK, and T cells were treated with 10 µM AZA for 24 h, the expression level of ICN1 and ICN2 was validated by western blot. (E) The expression of Notch1 and Notch2 in mouse spleen measured by ICH. IHC, immunohistochemistry.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Mass Spectrometry, Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Azelaic acid (AZA) exerts anti-leukemic effect by activating the Notch signaling pathway. (A) Notch responsive elements were transfected into 293T cells after 24 h. Cells were then treated with 10 µM AZA, 10 µM RO4929097, and combination for 24 h, the Notch signaling reporter assay was measured by dual luciferase reporter activity. (B) Validation of the RNA expression of Notch1 and Notch2, the downstream target genes HES1 and HEY1 in Molm-13 and THP-1 cells by qPCR. (C) The protein expression level of ICN1, ICN2, HEY1, and HES1 in acute myeloid leukemia (AML) cell lines after treatment of AZA and RO4929097 and their detection by western blot. ImageJ was used for the densitometric analysis. Data represent means ± SD. (D) Molm-13 cells were pretreated with 10 µM RO4939097 for 24 h, and then treated with 10 µM AZA for another 24 h. Cells were collected for apoptosis analysis. (E) NK cells and T cells were pretreated with 10 µM AZA, 10 µM RO4929097, and combination for 24h before co-culture with THP-1 cell and Molm-13 cells at an E:T ratio of 5:1. The cytotoxicity of NK and T was determined by detecting the LDH release rate. (F) NK and T cells were pre-treated with AZA and RO4929097, then co-cultured with THP-1 cell at an E:T ratio of 3:1 for 4 h. The level of TNF-α and IFN-γ in the supernatant was measured by ELISA. A Total of three independent experiments were performed. *P < 0.05, **P < 0.01,***P < 0.001.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Transfection, Reporter Assay, Luciferase, Activity Assay, Biomarker Discovery, RNA Expression, Expressing, Western Blot, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling

doi: 10.1084/jem.20220342

Figure Lengend Snippet: Increased p-CD19 levels in β1 KO transitional and MZ B cells and list of deregulated genes in transitional B cells treated with PI3K inhibitor. Related to and . (A) Transitional and MZ B cells from β1 WT and β1 KO mice were stimulated for different time points with anti-mouse IgM F(ab′)2 antibody and the phosphorylation of CD19 was evaluated by flow cytometry. (B) Mean (±SD) of phosphorylation of CD19 MFI is plotted. (A and B) n = 3 mice. Data are representative of three different experiments; significance is calculated by ANOVA test (* P < 0.05, *** P < 0.001). (C and D) Selected list of key genes from functional classification of up- (red) and downregulated (green) genes in PI3K inhibitor–treated β1 WT (C) and β1 KO (D) transitional B cells. RNA-seq was performed once. (E) Lysates of transitional β1 WT and β1 KO B cells were incubated with beads cross-linked with α-Grb2 or control α-Ig antibodies. Samples were washed and resolved by SDS-PAGE. Grb2 and FAK were detected by immunoblot analysis with specific antibodies. Data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: For Notch2, ILK, Grb2, and FAK immunoblots, cells were lysed as detailed above and samples were blotted with Notch2 antibody (D76A6; rabbit monoclonal; Cell Signaling Technology), ILK antibody (4G9; rabbit monoclonal; Cell Signaling Technology), and Grb2 antibody (Y237; rabbit monoclonal; Abcam), respectively, and Tubulin-HRP (Mouse IgG2b, Proteintech).

Techniques: Phospho-proteomics, Flow Cytometry, Functional Assay, RNA Sequencing, Incubation, Control, SDS Page, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling

doi: 10.1084/jem.20220342

Figure Lengend Snippet: Grb2 adaptor interacts with ILK in β1 KO transitional B cells. (A) Analysis of the expression by Western blot of ILK and Grb2 in β1 WT and β1 KO transitional B cells. Tubulin expression was used as a loading control. Each line represents a different mouse. A quantification of ILK (left) and Grb2 (right)/loading control ratio after normalization is shown for independently performed experiments. The mean ± SD is plotted. Co-IP to detect the association of Grb2 with ILK. (B and C) Lysates of β1 WT and β1 KO Fo (B) and transitional (C) B cells were incubated with beads cross-linked with α-ILK or control α-Ig antibodies. Samples were washed and resolved by SDS/PAGE. Grb2 and ILK were detected by immunoblot analysis with specific antibodies. A quantification of the Grb2/ILK ratio for transitional B cells is shown for independently performed experiments. Co-IPs with α-ILK and control α-Ig antibodies are represented by black and red dots, respectively. The mean ± SD is plotted. (A–C) n = 3 mice. Each circle in the graphs represents data from one mouse. Data are representative of three independent experiments. Mean and SD are indicated by horizontal lines in the data points. Source data are available for this figure: .

Article Snippet: For Notch2, ILK, Grb2, and FAK immunoblots, cells were lysed as detailed above and samples were blotted with Notch2 antibody (D76A6; rabbit monoclonal; Cell Signaling Technology), ILK antibody (4G9; rabbit monoclonal; Cell Signaling Technology), and Grb2 antibody (Y237; rabbit monoclonal; Abcam), respectively, and Tubulin-HRP (Mouse IgG2b, Proteintech).

Techniques: Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Incubation, SDS Page

Journal: The Journal of Experimental Medicine

Article Title: Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling

doi: 10.1084/jem.20220342

Figure Lengend Snippet: Increased p-CD19 levels in β1 KO transitional and MZ B cells and list of deregulated genes in transitional B cells treated with PI3K inhibitor. Related to and . (A) Transitional and MZ B cells from β1 WT and β1 KO mice were stimulated for different time points with anti-mouse IgM F(ab′)2 antibody and the phosphorylation of CD19 was evaluated by flow cytometry. (B) Mean (±SD) of phosphorylation of CD19 MFI is plotted. (A and B) n = 3 mice. Data are representative of three different experiments; significance is calculated by ANOVA test (* P < 0.05, *** P < 0.001). (C and D) Selected list of key genes from functional classification of up- (red) and downregulated (green) genes in PI3K inhibitor–treated β1 WT (C) and β1 KO (D) transitional B cells. RNA-seq was performed once. (E) Lysates of transitional β1 WT and β1 KO B cells were incubated with beads cross-linked with α-Grb2 or control α-Ig antibodies. Samples were washed and resolved by SDS-PAGE. Grb2 and FAK were detected by immunoblot analysis with specific antibodies. Data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: For Notch2, ILK, Grb2, and FAK immunoblots, cells were lysed as detailed above and samples were blotted with Notch2 antibody (D76A6; rabbit monoclonal; Cell Signaling Technology), ILK antibody (4G9; rabbit monoclonal; Cell Signaling Technology), and Grb2 antibody (Y237; rabbit monoclonal; Abcam), respectively, and Tubulin-HRP (Mouse IgG2b, Proteintech).

Techniques: Phospho-proteomics, Flow Cytometry, Functional Assay, RNA Sequencing, Incubation, Control, SDS Page, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling

doi: 10.1084/jem.20220342

Figure Lengend Snippet: Grb2 adaptor interacts with ILK in β1 KO transitional B cells. (A) Analysis of the expression by Western blot of ILK and Grb2 in β1 WT and β1 KO transitional B cells. Tubulin expression was used as a loading control. Each line represents a different mouse. A quantification of ILK (left) and Grb2 (right)/loading control ratio after normalization is shown for independently performed experiments. The mean ± SD is plotted. Co-IP to detect the association of Grb2 with ILK. (B and C) Lysates of β1 WT and β1 KO Fo (B) and transitional (C) B cells were incubated with beads cross-linked with α-ILK or control α-Ig antibodies. Samples were washed and resolved by SDS/PAGE. Grb2 and ILK were detected by immunoblot analysis with specific antibodies. A quantification of the Grb2/ILK ratio for transitional B cells is shown for independently performed experiments. Co-IPs with α-ILK and control α-Ig antibodies are represented by black and red dots, respectively. The mean ± SD is plotted. (A–C) n = 3 mice. Each circle in the graphs represents data from one mouse. Data are representative of three independent experiments. Mean and SD are indicated by horizontal lines in the data points. Source data are available for this figure: .

Article Snippet: For Notch2, ILK, Grb2, and FAK immunoblots, cells were lysed as detailed above and samples were blotted with Notch2 antibody (D76A6; rabbit monoclonal; Cell Signaling Technology), ILK antibody (4G9; rabbit monoclonal; Cell Signaling Technology), and Grb2 antibody (Y237; rabbit monoclonal; Abcam), respectively, and Tubulin-HRP (Mouse IgG2b, Proteintech).

Techniques: Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Incubation, SDS Page