Journal: International Journal of Molecular Sciences

Article Title: Renalase Protects against Renal Fibrosis by Inhibiting the Activation of the ERK Signaling Pathways

doi: 10.3390/ijms18050855

Figure Lengend Snippet: Renalase inhibits the TGF-β1-mediated increase of phosphorylated ERK1/2 in vitro. ( A ) Human proximal tubular epithelial cells (HK-2) were treated with 2 ng/ml TGF-β1 in the presence or absence of renalase (1000 ng/mL) for 48 h. Western blotting revealed that renalase inhibited TGF-β1-induced ERK1/2 phosphorylation, and did not affect the phosphorylation of Smad2/3 and p38. Results are presented as percentages of control values after normalization to GADPH and are the means ± SD of three independent experiments. * p < 0.05, compared with control groups; # p < 0.05, compared with TGF-β1-stimulated groups; NS: not statistically significant compared with control groups. n = 5 per group; ( B ) Changes of TGF-β1 signaling pathway in UUO rats. Renalase inhibited ERK1/2 phosphorylation and did not affect the phosphorylation of Smad2/3 and p38. Results are presented as percentages of control values after normalization to GADPH and are the means ± SD of three independent experiments. * p < 0.05, compared with sham + Ad-b-gal groups; # p < 0.05, compared with UUO + Ad-renalase groups; n = 5 per group; ( C ) Human proximal tubular epithelial cells (HK-2) were treated with 2 ng/mL TGF-β1 in the presence or absence of renalase (1000 ng/mL) or PD98059 (20 μM) for 48 h. Western blotting showed that renalase has a similar effect with PD98059 to minimize the increase of FN, Col-I, α-SMA and ERK1/2 phosphorylation and restore the decrease of e-cadherin induced by TGF-β1. While renalase and PD98059 were given at the same time to TGF-β1-stimulated cells, the protective effect of renalase and PD98059 was not superimposed. Results are presented as percentages of control values after normalization to GADPH and are the means ± SD of three independent experiments. * p < 0.05, compared with control groups; # p < 0.05, compared with TGF-β1-stimulated groups; NS: not statistically significant between groups. n = 5 per group.

Article Snippet: Then, a plasmid containing human ERK1/MAPK3 (RC204196, Origene, Rockville, MD, USA) was transfected into the cells with Lipofectamine ® 3000 Transfection Reagent (Invitrogen, Burlington, ON, Canada) following the manufacturer’s protocol.

Techniques: In Vitro, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Renalase Protects against Renal Fibrosis by Inhibiting the Activation of the ERK Signaling Pathways

doi: 10.3390/ijms18050855

Figure Lengend Snippet: The role of ERK signaling pathway in the inhibition of EMT by renalase. ( A ) Results of plasmid transfection to overexpress ERK1. The transfection efficiency was observed with different doses of transfection reagents: low dose and high dose. Compared with control groups, the expression of p-ERK1/2 in the plasmid transfection groups were significantly higher than controls and the expression in the high dose group was higher than in the low dose group at 48 and 72 h. * p < 0.05, compared with control groups; # p < 0.05, compared with low dose groups. C: control group; L: low dose group; H: high dose group; NS: not statistically significant; ( B ) Effect of renalase on EMT and fibrosis induced by TGF-β1 in transfected cells. HK-2 were treated with plasmid and high dose transfection reagents in the presence or absence of TGF-β1 (2 ng/mL) and renalase (1000 ng/mL) as indicated for 48 h. Western blotting demonstrated that renalase cannot inhibit the increased expression of α-smooth muscle actin, collagen-I, and fibronectin and reverse the decreased expression of E-cadherin induced by TGF-β1 when ERK is overexpressed. * p < 0.05, compared with control groups; NS: not statistically significant between groups; ( C ) Effect of renalase and PD98059 on EMT and fibrosis induced by TGF-β1 in transfected cells. HK-2 were treated with plasmid and high dose transfection reagents in the presence or absence of TGF-β1 (2 ng/mL) and renalase (1000 ng/mL) or PD98059(20 µM) as indicated for 48 h. Western blotting demonstrated that the expression levels of p-ERK1/2 between groups were similar and that neither renalase nor PD98059 can inhibit the increased expression of α-smooth muscle actin, collagen-I, fibronectin or reverse the decreased expression of E-cadherin induced by TGF-β1 when ERK is overexpressed. * p < 0.05, compared with control groups. Results are presented as percentages of control values after normalization to GADPH and are the means ± SD of three independent experiments; n = 3 per group.

Article Snippet: Then, a plasmid containing human ERK1/MAPK3 (RC204196, Origene, Rockville, MD, USA) was transfected into the cells with Lipofectamine ® 3000 Transfection Reagent (Invitrogen, Burlington, ON, Canada) following the manufacturer’s protocol.

Techniques: Inhibition, Plasmid Preparation, Transfection, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in microglia induces cell proliferation and activation through ERK. Primary microglia cells were prepared from C57BL/6J embryos. Cells were transduced with lentiviral vector, BRAF WT and BRAF V600E for 24 hours and then cultured in DMEM/F12 for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B, C) Cells were transfected with control-siRNA, or si-JNK, or si-ERK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for ERK and JNK 48 hours after viral transduction and quantified expression levels normalized to GAPDH. (D) Cells were transfected with control-siRNA, or si-JNK, or si-ERK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for BRAF-related signaling proteins and quantified expression levels normalized to GAPDH. (E, F) Immunostaining for Iba1 and Ki67, Iba1 + cell counts, and percentage of Ki67 + microglia (scale bar: 100 μm), and quantitative morphological analyses (percentage of ameboid-like microglia cells, length, area, length to area ratio in cells without and with siRNA transfection (scale bar: 50 μm). Iba1 (green, astrocytes), DAPI (blue, nuclei), Ki67 (red, proliferative cells). (G, H) Flow cytometry analysis of cell cycle. (I) MTS cell viability assay at 48, 72, 96 and 120 hours following viral transduction. (J) NO release in culture media by Griess reaction. (K) qPCR analysis of inflammatory and antioxidant markers in cells normalized to GAPDH. (L) IL-1β, IL-6 and TNF-α levels in culture medium measured by ELISA. Data are represented as mean ± SEM, n = 9. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates under each condition. ERK: Extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; NO: nitric oxide; qPCR: quantitative polymerase chain reaction.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Activation Assay, Transduction, Plasmid Preparation, Cell Culture, Western Blot, Transfection, Control, Immunostaining, Flow Cytometry, Viability Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: Conditioned medium from BRAF V600E -expressing microglial cells but not astrocytes induces neuronal cell death. Primary cortex neurons were prepared from C57BL/6J embryos and cultured for 5 days, and the original medium was then replaced with conditional medium for 72 hours. (A, B) Immunostaining for MAP2 (scale bar: 50 μm) and MAP2 + cell counting. (C) LDH release in neurons treated with conditioned medium from primary astrocytes transduced with lentiviral BRAF vectors. (D–F) Immunostaining for MAP2 (scale bar: 50 μm; D) and MAP2 + cell counting in neurons treated with conditioned medium from primary microglia transduced with lentiviral BRAF vectors with and without siRNA transfection (E, F). (G, H) LDH release from neurons treated with conditioned medium from primary microglia transduced with lentiviral BRAF vectors with and without siRNA transfection. * P < 0.05, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. MAP2 (green, neurons), DAPI (blue, nuclei) in A and D. DAPI: 4′,6-Diamidino-2-phenylindole; LDH: lactate dehydrogenase; MAP2: microtubule-associated protein 2.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Immunostaining, Cell Counting, Transduction, Transfection

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in neurons promotes cell death through the JNK pathway. Primary cortex neurons were prepared from C57BL/6J embryos. Cells were cultured for 5 days, transduced with lentiviral vector, BRAF WT and BRAF V600E for 24 hours, and then cultured in NB-A for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B) qPCR analysis c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (C–E) Immunostaining for MAP2 (scale bar: 50 μm), MAP2 + cell counting, and LDH release. (F) Primary cortex neurons were transfected with control-siRNA or si-JNK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (G) qPCR analysis of c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (H) Immunostaining for MAP2 (sale bar: 50 μm), (I) MAP2 + cell counting, (J) and LDH release. Primary cortex neurons were transfected with control-siRNA or si-ERK for 24 hours before transduction with BRAF viral vectors. (K) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (L) qPCR analysis of c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (M–O) Immunostaining for MAP2 (scale bar: 50 μm), MAP2 + cell counting, and LDH release. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. MAP2 (green, neurons), DAPI (blue, nuclei) in C, H, and M. DAPI: 4′,6-Diamidino-2-phenylindole; ERK: extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; LDH: lactate dehydrogenase; MAP2: microtubule-associated protein 2; NB-A: neurobasal media; qPCR: quantitative polymerase chain reaction; TNF-α: tumor necrosis factor-alpha; WT: wide type.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, Western Blot, Immunostaining, Cell Counting, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin suppresses either BRAF V600E or RAS mutant cell growth in CDX model. a NOD-SCID mice were injected subcutaneously with SK-MEL-2 (NRAS mut, 5 × 10 6 cell/mouse), A375 (BRAF V600E, 1 × 10 7 cell/mouse), SK-MEL-28 (BRAF V600E, 5 × 10 6 cell/mouse) and HCT116 (KRAS mut, 1 × 10 7 cell/mouse) cells mixed with Matrigel (1:1); erianin (50 mg/kg), vemurafenib (50 mg/kg) or the combine was given through oral gavage and the size of the tumors was monitored twice per week. Tumor volume (mm 3 ) = (length × width × height) × 0.52. SK-MEL-2: n = 10; A375 and SK-MEL-28: n = 8; HCT116: n = 9. b The photographs show tumors from CDX mice treated with vehicle, erianin, vemurafenib, or the combination. c The weight of the tumors was quantified and expressed as the treatment groups compared with the vehicle-treated group. Data were presented as mean ± S.D. One-way ANOVA test. * p < 0.05; ** p < 0.01. d Western blotting shows the expression of phospho-MEK1/2 and phospho-ERK1/2 by erianin in SK-MEL-2, A375, and SK-MEL-28 CDX tumor tissues. The tissue lysates were prepared from CDX tumor tissues in each treatment group. Three samples were randomly prepared for each group and every blot shows one sample. e The quantization (IOD values) of IHC staining in the treatment groups compared with the vehicle-treated group. Each point represents the IOD values of four quantified data from one mouse. Scale bars: 50 μm. One-way ANOVA test. *** p < 0.001. f Kaplan–Meier curve depicting tumors less than 1000 mm 3 in the treatment groups compared with the vehicle-treated group

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, Injection, Western Blot, Expressing, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin exerts antitumor efficacy in melanoma and colorectal cancer in vivo. a Tumor pharmacodynamic assay was performed in tumor-bearing NPG mice (tumor has been passaged from melanoma patient to mice for three generations). The photographs show tumors from melanoma PDX mice treated with vehicle or drugs. b The effect of erianin on the volume of PDX tumors over time (within 78 days) was plotted. Vehicle, erianin (50 mg/kg, once a day), vemurafenib (50 mg/kg, once a day), erianin and vemurafenib combination therapy, cobimetinib (5 mg/kg, twice a week) or vemurafenib and cobimetinib combination therapy (once a day and twice a week, respectively) were administered by oral gavage, n = 8 in each group. Tumor volume was measured once a week. One-way ANOVA test. * p < 0.05; ** p < 0.01. c Tumor weight was measured after treatment on the last day of the study. d The expression of phospho-MEK1/2 and phospho-ERK1/2 were examined by immunofluorescence analysis. Scale bars: 20 μm. One-way ANOVA test. *** p < 0.001. e Antitumor efficacy of erianin with or without immunity using B16F10 cell xenograft in C57BL-6J mouse. f , g Trend of tumor volume over time and tumor weight was measured after treatment on the last day of the study. One-way ANOVA test. * p < 0.05; ** p < 0.01. h The model depicts that erianin suppresses constitutive activation of MAPK signaling pathway in either BRAF V600E or RAS mutant cancers (Created with BioRender.com). Through inhibition of CRAF and MEK1/2 kinases, erianin suppresses phospho-MEK1/2 and phospho-ERK1/2 without paradoxical activation in vitro and in vivo

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: In Vivo, Expressing, Immunofluorescence, Activation Assay, Mutagenesis, Inhibition, In Vitro