Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Activation of Notch3 in HIV-1-associated nephropathy. (A-D) Immunolabeling was performed for the presence of Notch3 in renal paraffin sections. Notch3 (green) labeling in kidney sections of 3-month-old wild-type (WT) (A,C) and HIV-Tg26 (Tg) (B,D) mice is shown. Arrows indicate glomerular and tubular interstitial cells highly positive for Notch3 expression. Asterisks indicate blood vessels in which Notch3 is normally expressed. (E,F) Quantification of glomerular (E) and tubular (F) Notch3 expression (intensity, green) as assessed using ImageJ. (G,H) Kidney biopsy from an unaffected individual (G) versus kidney biopsy from a patient with HIV-1-associated nephropathy (HIVAN) (H) (representative of n =3 in each group), showing nuclear NOTCH3 expression (arrows), indicating activation. Asterisks indicate blood vessels with bright Notch3 expression. Dashed line outlines glomerulus with no Notch3 expression. (I) Quantification of NOTCH3 expression (intensity, green) in patients with HIVAN and unaffected individuals (NHK), as assessed using ImageJ. Unpaired two-tailed Student's t -test was used; data are presented as percentage area positive for green labeling. All experiments were performed in triplicates. * P <0.05, ** P <0.01, **** P <0.0001. Scale bars: 50 µm.

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques: Activation Assay, Immunolabeling, Labeling, Expressing, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Notch3 deletion improves disease progression and lifespan in HIV-Tg26 mice. (A) Kaplan–Meier curve showing the 6 months mortality rate in HIV-Tg26 (Tg) mice ( n =26) and HIV-Tg26 with Notch3 knocked out (Tg-N3KO) mice ( n =28). (B) Phenotypic appearance of WT, WT with Notch3 knocked out (N3KO), Tg and Tg-N3KO mice; note the skin papillomata on the forehead of the Tg mouse, whereas the Tg-N3KO mouse appears normal. (C,D) Skin lesions/mouse were quantified ( n =9 each), and area per lesion was measured and expressed as surface area per cm 2 . Unpaired two-tailed Student's t -test was used for statistical analysis. (E) Urine was collected in metabolic cages overnight from WT, N3KO, Tg and Tg-N3KO mice at 3 months of age before euthanasia. Proteinuria was assessed in 2 µl urine by SDS-PAGE followed by Coomassie Blue staining of the gels. Bovine serum albumin (BSA) was used as a positive control. (F) Albumin and creatinine ratio in urine was measured using ELISA ( n =4, WT; n =4, N3KO; n =10, Tg; n =10, Tg-N3KO) and expressed as µg albumin/mg creatinine. One-way ANOVA. (G) Renal function was also assessed in serum using a blood urea nitrogen (BUN) assay kit, and results are expressed as BUN mg/dl ( n =4, WT; n =4, N3KO; n =6, Tg; n =7,Tg-N3KO). One-way ANOVA. * P <0.05, *** P <0.001.

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques: Biomarker Discovery, Two Tailed Test, SDS Page, Staining, Positive Control, Enzyme-linked Immunosorbent Assay, Nitrogen Bun Assay

Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Notch3 deletion ameliorates kidney injury in HIV-Tg26 mice. Kidney sections from 3-month-old WT, N3KO, Tg and Tg-N3KO mice were stained with periodic acid–Schiff (PAS) to determine kidney injury. (D-F) PAS staining indicated tubulointerstitial injury (D), glomerular tubular injury (E) and inflammation (F). Note the severe kidney injury in Tg kidneys ( n =9) compared to Tg-N3KO kidneys ( n =12). Arrows in D indicate protein casts; arrows in E indicate comparisons between normal-looking (N3KO), Tg26 and Tg-N3KO glomeruli, showing the extent of sclerosis. (A-C) Quantitation of percentage tubulointerstitial injury (A), percentage glomerular injury (B) and percentage infiltration (C). Unpaired two-tailed Student's t -test was used for statistical analysis. ns, not significant; * P <0.05. Scale bar: 100 µm.

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques: Staining, Quantitation Assay, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Genes downregulated in Tg-N3KO and Tg-N4KO mice compared to Tg26 mice

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Notch3 targets HIV-1 activity. (A) Heatmap showing differential expression of genes obtained from mRNA sequencing of kidneys from 3-month-old WT, N3KO, Tg and Tg-N3KO mice. Note the asterisks for nef and env . (B,C) Quantitative PCR (qPCR) validating nef (B) and env (C) ( n =5-7), followed by unpaired two-tailed Student's t -test for statistical analysis. (D) qPCR to compare Tg-N3KO and Tg-N4KO in repressing nef ( n =3 per condition). One-way ANOVA followed by Tukey's test was used for statistical analysis. (E) HIV-long terminal repeat (LTR) promoter luciferase construct and Notch3 intracellular domain (N3IC) or PCDNA3.1 [empty vector (EV)] were transiently transfected into podocytes, followed by promoter reporter luciferase assays. Data were averaged from eight assays in three independent experiments. Vector-transfected control in each experiment was set to one, and relative fold change in LTR promoter activity was calculated. (F) Podocytes were transfected with HIV-LTR construct (PNL4) or EV. After 24 h, lysates were subjected to western blot analysis of N3IC and β-actin. Data were normalized to β-actin via ImageJ and presented as fold change ( n =4). Unpaired two-tailed Student's t -test was used for statistical analysis. * P <0.05, **** P <0.0001.

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques: Activity Assay, Quantitative Proteomics, Sequencing, Real-time Polymerase Chain Reaction, Two Tailed Test, Luciferase, Construct, Plasmid Preparation, Transfection, Control, Western Blot

Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Notch3 targets immune cell infiltration. (A-C) Volcano plots comparing gene expression in kidneys from Tg versus WT (A), Tg-N3KO versus WT (B), and Tg versus TG-N3KO (C) mice. (D) qPCR for Ccl2 expression. One-way ANOVA followed by Tukey's test was used for statistical analysis. (E) Immunohistochemistry (IHC) for MMP10 and NFκB (p65) in renal sections of WT, N3KO, Tg and Tg-N3KO mice. Arrows show glomerular and tubular areas with high expression. Scale bar: 100 µm. (F) Paraffin sections from kidneys of WT, N3KO, Tg and Tg-N3KO mice, labeled for immune cell markers CD68, FOXP3 and CD8. Arrow indicates CD68 + macrophages found in clumps in Tg kidney. Scale bar: 50 µm. (G) Quantification of CD68 + , FOXP3 + and CD8 + HIVAN cells from Tg and Tg-N3KO mice, assessed in Tg ( n =9) and Tg-N3KO ( n =9) kidneys unaware of experimental group. Each dot represents the percentage of positive cells from the entire kidney where inflammatory invasions were prominent. Unpaired two-tailed Student's t -test was used for statistical analysis. (H) Serial sections from a kidney biopsy from a patient with HIVAN immunostained for the presence of CD68 and NOTCH3. Note the presence of CD68 + cells in and around glomerulus; the same areas were also labeled brightly for NOTCH3 (arrows). Scale bar: 100 µm. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques: Gene Expression, Expressing, Immunohistochemistry, Labeling, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease

doi: 10.1242/dmm.052056

Figure Lengend Snippet: Notch3 deletion targets systemic inflammation in HIV-Tg26 mice. (A) Schematic showing macrophage isolation from bone marrow in mice. (B) Labeling of differentiated bone marrow cells expressing the macrophage marker F4/F80 in fixed cells. Scale bar: 50 µm. (C) Lysates obtained from differentiated macrophages of WT and Tg mice were subjected to western blot analysis for N3IC, Dll4 and jagged 1 (J1). CD68 was used as a loading control. Ponceau S provides an additional loading control. (D-F) Quantification of N3IC (D), Dll4 (E) and jagged 1 (F) in bone marrow cells obtained from three mice in each group ( n =3). Unpaired two-tailed Student's t -test was used for statistical analysis. (G,H) ELISA was performed for the presence of TNFα (TNF; G) and MCP-1 (H) in serum obtained from 3-month-old WT, N3KO, Tg and Tg-N3KO mice ( n =6-12), both males and females. Data are expressed in pg/ml. One-way ANOVA followed by Tukey's test was used for statistical analysis. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Notch3 d1 (N3KO) mice were obtained from The Jackson Laboratory ( JAX:023807 ).

Techniques: Isolation, Labeling, Expressing, Marker, Western Blot, Control, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: Prediction of target molecule for miRNA-206. Note: A, Starbase V2.0 online database prediction for the binding site of mirNA-206 at the 3 ’ UTR of Notch3; B, Dual luciferase reporter gene assay for the regulation of miRNA-206 on Notch3; C, qRT-PCR for the regulation of miRNA-206 on Notch3. Notch3 wild type: Notch3-3 ′ UTR WT; Notch3 mutant: Notch3-3 ’ UTR Mut; *P < 0.05; **P < 0.01, N=5.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Mutagenesis

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: Western blotting for expressions of Notch3 and BDNF. Note: A, Protein expression of Notch3 in different transected nerve groups; B, mRNA expression of Notch3 in different transected nerve groups; C, Detection on Notch3 for BDNF protein expression; D, Detection on Notch3 for mRNA expression of BDNF. Notch3 over-expression control group: Notch3-ctrl; Notch3 over-expression group: Notch3 mimic; Sham: Nerve exposed group not operated; Model: Denervation group; PAG: Nerve partial anastomosis group; AG: Nerve anastomosis group. *P < 0.05; **P < 0.01, N=5.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Western Blot, Expressing, Over Expression

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: miRNA-206 targeting Notch3 affects the recovery of neural function. Note: A: Western blotting for miRNA-206 regulating BDNF expression by Notch3; B-D: qRT-PCR for miRNA-206 regulating BDNF expression by Notch3; miRNA-206 over-expression control group: miRNA-206-ctrl, miRNA-206 over-expression group: miR-206 mimic; *P < 0.05; **P < 0.01, N=5.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Over Expression

Journal: Journal of Musculoskeletal & Neuronal Interactions

Article Title: MiRNA-206 Affects the Recovery of Sciatic Function by Stimulating BDNF Activity through the Down-regulation of Notch3 Expression

doi:

Figure Lengend Snippet: Plot of recovery mechanism of miRNA-206 affecting the recovery of neural function by targeting Notch3. Note: Over-expression of miRNA-206 inhibited Notch3 expression and activated the BDNF activity, which ultimately caused the recovery of traumatic sciatic function.

Article Snippet: The protein was then retransferred to PVDF membrane (Millipore, MA, USA, IPFL00010), and then closed with 5% skim milk and incubated with the following primary antibodies at 4°C: Mouse anti-Notch3 (1:1000, Millipore, MA, USA, APREST76106), Mouse anti-BNDF (1:1000, Sigma-Aldrich, USA, GF029) and Rabbit anti-GAPDH (1:1000, Millipore, MA, USA, G5262).

Techniques: Over Expression, Expressing, Activity Assay

Journal: British Journal of Pharmacology

Article Title: Costunolide represses hepatic fibrosis through WW domain‐containing protein 2‐mediated Notch3 degradation

doi: 10.1111/bph.14873

Figure Lengend Snippet: Costunolide (COS) suppressed the Notch3/HES1 pathway in vitro and in vivo. COS administration down‐regulated Notch3 especially NICD3 and HES1 protein expressions in BDL livers (a) and LX‐2 cells (b). (c) The nuclear and cytoplasmic extractions of LX‐2 cells were separated after COS treatment and analysed by western blot. (d) mRNA expressions of NOTCH3 and HES1 in LX‐2 cells. The mRNA/protein expression levels were normalized against GAPDH/GAPDH. (e) LX‐2 cells in dishes were fixed in 4% paraformaldehyde, stained with DAPI (blue) and Notch3 and HES1 (green), and imaged by confocal microscopy, Scale bar: 200 μm (up panel) and 100 μm (down panel). The values are expressed as the mean ± SD of five independent assays, # P < .05, significantly different from the control group, * P < .05, significantly different from the TGFβ1 treatment group in LX‐2 cells; ANOVA followed by Tukey's test

Article Snippet: Notch3 siRNA (SIGS0007282‐1), HES1 siRNA (SIGS0006714‐1) and WWP2 (SIGS0014969‐1) were acquired from RiboBio (Guangzhou, China).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Staining, Confocal Microscopy, Control

Journal: British Journal of Pharmacology

Article Title: Costunolide represses hepatic fibrosis through WW domain‐containing protein 2‐mediated Notch3 degradation

doi: 10.1111/bph.14873

Figure Lengend Snippet: Altering Notch3 expression influenced the antifibrotic activity of costunolide (COS). LX‐2 cells were transfected with (a) 2.5‐μg pcDNA3.1‐NICD3 or vector for 6 hr and subsequently treated with 2 ng·ml−1 TGFβ1 with or without different concentrations of COS for 24 hr after no FBS starvation. The protein expressions of NICD3, HES1 and liver fibrosis markers were determined by immunoblotting with the indicated antibodies. LX‐2 cells were transfected with (b) 50‐nM si‐Notch3‐1/2 or si‐Control and subsequently treated with 2 ng·ml−1 TGFβ1 with or without different concentrations of COS for 24 hr after no FBS starvation. The same protein expressions were detected by western blot with the indicated antibodies. GAPDH was used as a loading control for all western blot assays. The data are expressed as the mean ± SD of five independent assays, # P < .05, significantly different from the pcDNA3.1/si‐Control + COS 10‐μM group, * P < .05, significantly different from the pcDNA3.1‐NICD3 + 2T group. Δ P < .05, significantly different from the pcDNA3.1‐NICD3 + COS 10‐μM group; $ P < .05, significantly different from the si‐Notch3‐1 + 2T group. & P < .05, significantly different from the si‐Notch3‐2 + COS 10‐μM group. ANOVA followed by Tukey's test

Article Snippet: Notch3 siRNA (SIGS0007282‐1), HES1 siRNA (SIGS0006714‐1) and WWP2 (SIGS0014969‐1) were acquired from RiboBio (Guangzhou, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Control

Journal: British Journal of Pharmacology

Article Title: Costunolide represses hepatic fibrosis through WW domain‐containing protein 2‐mediated Notch3 degradation

doi: 10.1111/bph.14873

Figure Lengend Snippet: Altering HES1 expression influenced the antifibrotic activity of costunolide (COS). LX‐2 cells were transfected with (a) 2.5‐μg pcDNA3.1‐HES1 or vector. The protein expressions of Notch3‐ICD, HES1, and liver fibrosis markers were determined by immunoblotting with the indicated antibodies. LX‐2 cells were transfected with (b) 50‐nM si‐HES1‐1/2 or si‐Control. The same protein expressions were detected by western blot with the indicated antibodies. GAPDH was used as a loading control for all western blot assays. The data are expressed as the mean ± SD of five independent assays, # P < .05, significantly different from the pcDNA3.1/si‐Control + COS 10‐μM group; * P < .05, significantly different from the pcDNA3.1‐HES1 + 2T group and si‐Control + COS 10‐μM group; Δ P < .05, significantly different from the pcDNA3.1‐HES1 + COS 10‐μM group. $ P < .05, significantly different from the si‐HES1‐1 + 2T group; & P < .05, significantly different from the si‐HES1‐2 + COS 10‐μM group. ANOVA followed by Tukey's test

Article Snippet: Notch3 siRNA (SIGS0007282‐1), HES1 siRNA (SIGS0006714‐1) and WWP2 (SIGS0014969‐1) were acquired from RiboBio (Guangzhou, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Control

Journal: British Journal of Pharmacology

Article Title: Costunolide represses hepatic fibrosis through WW domain‐containing protein 2‐mediated Notch3 degradation

doi: 10.1111/bph.14873

Figure Lengend Snippet: Costunolide (COS) disrupted the interaction between WWP2 and PPM1G, which enhanced the effect of WWP2 on Notch3 degradation. The increase of Notch3 degradation further inhibited Notch3/HES1 pathway exerting repressive effect on liver fibrosis

Article Snippet: Notch3 siRNA (SIGS0007282‐1), HES1 siRNA (SIGS0006714‐1) and WWP2 (SIGS0014969‐1) were acquired from RiboBio (Guangzhou, China).

Techniques:

Journal: Oncotarget

Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

doi: 10.18632/oncotarget.9667

Figure Lengend Snippet: Cells were serum-starved overnight prior to treatment with TGF-β1 (200 pM) for the indicated duration. A. Western analysis of α-SMA levels. B. Immunofluorescent analysis of α-SMA expression. Green, α-SMA; blue, nuclei; bar size, 100 μm. C. Western analysis of p38, JNK1/2 and ERK1/2 phosphorylation. D. Western analysis of Notch3 and Notch1 levels. E. RT-PCR analysis of HES1 and HRT1 mRNA levels. The bands were quantified, normalized by β-actin or the respective unphosphorylated protein, and presented relative to 0 h (100%) as means ± SEM (n = 3-4). *, P < 0.05 vs 0 h.

Article Snippet: Scrambled and Notch3 siRNAs (5′-CCUGGCUACAA UGGUGAUATT-3′) were purchased from GenePharma (Shanghai, China).

Techniques: Western Blot, Expressing, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

doi: 10.18632/oncotarget.9667

Figure Lengend Snippet: Cells were serum-starved overnight prior to treatment with TGF-β1 (200 pM). A-B. Effect of pretreatment with p38 inhibitor SB203580 (10 μM, 1 h) and JNK inhibitor SP600125 (20 μM, 1 h) on p38 and JNK1/2 phosphorylation and α-SMA level after TGF-β1 treatment. C. Impact of Notch3 siRNA knockdown or inhibitor DAPT (10 μM, 1 h) on TGF-β1-induced α-SMA expression. Cells were transfected with a scrambled control (siCtrl) or Notch3 siRNA (siNotch3) for 24 h prior to treatment with TGF-β1 for 48 h. D. Effect of DAPT pretreatment (10 μM, 1 h) on TGF-β1 (1 h)-induced p38 and JNK1/2 activation, as well as ERK1/2 activity. E. Effect of p38 inhibitor SB203580 (10 μM, 1 h), JNK inhibitor SP600125 (20 μM, 1 h) and ERK inhibitor U0126 (10 μM, 1 h) on Notch3 expression after TGF-β1 treatment for 48 h. The protein levels of α-SMA and Notch3 were quantified, normalized by β-actin and presented relative to the one treated with TGF-β1 alone (100%) as means ± SEM (n = 3-4). *, P < 0.05.

Article Snippet: Scrambled and Notch3 siRNAs (5′-CCUGGCUACAA UGGUGAUATT-3′) were purchased from GenePharma (Shanghai, China).

Techniques: Phospho-proteomics, Knockdown, Expressing, Transfection, Control, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

doi: 10.18632/oncotarget.9667

Figure Lengend Snippet: A. Induction of reactive oxygen species (ROS) by TGF-β1. IMR-90 fibroblasts were treated with TGF-β1 (200 pM, 1 h), with or without NAC or catalase pre-treatment, followed by incubation with 2′,7′-dichlorodihydrofluorescein diacetate (DCF, 10 μM, 15 min). Representative images of DCF fluorescence were shown, and ROS production was quantified and presented relative to that without any treatment as means ± SEM (n = 3; *, P < 0.05). Bar size, 100 μm. B-E. Western analyses of the effect of antioxidant pre-treatment on basal and TGF-β1-induced expression of α-SMA (B), Notch3 (C), phospho-p38 (D), and phospho-JNK1/2 (E). Cells were pretreated with NAC (4 mM, 1 h) or catalase (1000 U/L, 4 h) prior to TGF-β1 (200 pM) stimulation for 48 h (for α-SMA and Notch3) or 1 h (for p38 and JNK1/2). The expression levels were quantified and normalized with their respective controls, and presented relative to that treated with TGF-β1 alone as means ± SEM (n = 3-4; *, P < 0.05). F-G. Expression of phosphorylated ERK1/2, p38, and JNK1/2 in IMR-90 fibroblasts treated with NAC (F, 4 mM) or catalase (G, 1000 U/L) for 0-240 min.

Article Snippet: Scrambled and Notch3 siRNAs (5′-CCUGGCUACAA UGGUGAUATT-3′) were purchased from GenePharma (Shanghai, China).

Techniques: Incubation, Fluorescence, Western Blot, Expressing

Journal: Oncotarget

Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

doi: 10.18632/oncotarget.9667

Figure Lengend Snippet: A. Representative images of H&E staining and TGF-β1 expression determined by immunohistochemistry. Bar size, 100 μm. B. Western blot analyses of p38, JNK1/2 and ERK1/2 phosphorylation, α-SMA and Notch3 expression. The protein bands were quantified, normalized with their non-phosphorylated counterparts or β-actin, and presented relative to that of saline-treated controls as means ± SEM (n = 3-4). *, P < 0.05 vs saline. #, P < 0.05 vs day 7.

Article Snippet: Scrambled and Notch3 siRNAs (5′-CCUGGCUACAA UGGUGAUATT-3′) were purchased from GenePharma (Shanghai, China).

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Phospho-proteomics, Saline

Journal: Neoplasia (New York, N.Y.)

Article Title: Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer.

doi: 10.1016/j.neo.2017.08.006

Figure Lengend Snippet: Figure 6. Human claudin-low breast cancer exhibits low levels of LFNG and HES1 gene expression. (A) Boxplots for expression values of LFNG, NOTCH3, JAG1, HES1 and HEY1 in 6 subtypes of human breast cancer (dataset GSE18229). (B) Heat map for expressions of selected Notch pathways genes in 6 subtypes of human breast cancer (METABRIC dataset). (C) Boxplots for expression values of LFNG, NOTCH3, JAG1, HES1 and HEY1 in 6 subtypes of human breast cancer (METABRIC dataset). Basal: Basal-like. Claudin: Claudin-low. Her2: HER2-positive. LumA: Luminal A. LumB: Luminal B. Normal: Normal breast-like. p values were calculated by comparing expression means across all subtypes.

Article Snippet: Primary antibodies used for immunostaining were: Notch1 (Cell Signaling, No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, 55,114–1-AP, 1:100), E-cadherin (Cell Signaling, No. 3195, 1:100), Vimentin (Cell Signaling, No. 5741, 1:100), Twist (Santa Cruz, sc-6070, 1:100), PDGFRα (Santa Cruz, sc-338, 1:100), Cytokeratin 8 (Santa Cruz, sc-101,459, 1:200), Cytokeratin 14 (Santa Cruz, sc-53,253, 1:200), Ki67 (Abcam, ab16667, 1:200), and Lfng (Abcam, ab192788, 1:100).

Techniques: Gene Expression, Expressing