Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: The expression of Raf1 in AgRP neurons of DIO mice was significantly elevated. A and B: Western blotting analysis of the protein levels of RAF1 in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice). C: Representative FISH staining shows co-localization of Raf1 and Agrp mRNA in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice; scale bars, 100 μm). D: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in AgRP neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 149; HFD, N = 96). E: Representative FISH staining shows co-localization of Raf1 and Pomc mRNA in the hypothalamus of mice fed an NCD or an HFD (scale bars, 100 μm). Arrows in C and E indicate the co-localization of the genes. F: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in POMC neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 166; HFD, N = 176). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05 and *** P < 0.001 by unpaired Student's t -tests and non-parametric tests (B, D, and F). Abbreviations: AgRP, agouti-related peptide; DIO, diet-induced obesity; FISH, fluorescence in situ hybridization; HFD, high-fat diet; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; POMC, pro-opiomelanocortin.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Expressing, Western Blot, Staining, Fluorescence, In Situ Hybridization

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Overexpression of Raf1 in AgRP neurons promoted obesity and related metabolic disorders. A: Schematic diagram of bilateral injections of AAV-DIO- Raf1 -HA and its control AAV-DIO-mCherry into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. B: Representative IF staining of mCherry and RAF1-HA in AgRP neurons ( n = 3 mice; scale bars, 100 μm). C: Representative image of control and AgRP- Raf1 -OE mice fed an NCD. D–L: Various metabolic indicators, including body weight gain curves ( n = 8–9 mice; D), food intake ( n = 8–9 mice; E), body mass ( n = 6–7 mice; F), representative H&E staining images of liver, BAT, and WAT ( n = 3 mice; scale bars: 100 μm; G), droplet area of WAT ( n = 3 mice; H), respiratory exchange ratio ( n = 4 mice; I), energy expenditure ( n = 4 mice; J), GTT ( n = 6 mice; K), and serum insulin and leptin levels ( n = 4–6 mice; L) in both control and AgRP- Raf1 -OE mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (E, H, and L), two-way ANOVA with Bonferroni's post hoc test (D and K), and multiple t -tests (F, I, and J). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E: hematoxylin and eosin; IF, immunofluorescence; NCD, normal chow diet; NPY, neuropeptide Y; ns, not significant; rAAV-DIO- Raf1 /mCherry, recombinant adeno-associated virus encoding Raf1 -HA or mCherry; RAF1, v-raf-leukemia viral oncogene 1; OE, overexpression; WAT, white adipose tissue.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Over Expression, Control, Staining, Immunofluorescence, Recombinant, Virus

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Slight promotion of obesity development was observed in AgRP- Raf1 -OE mice under HFD feeding. A: Representative image of control and AgRP- Raf1 -OE mice fed an HFD. B–J: Various metabolic indicators including body weight gain curves ( n = 10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -OE mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05 and ** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; ns, not significant; OE, overexpression; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Control, Staining, Over Expression

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Screening and in vivo validation of sgRNA. A: Gel electrophoresis shows the cleavage effect of sgRNA targeting the Raf1 gene. B: The schematic diagrams show sgRNA1 and sgRNA2. C and D: Western blotting analysis of RAF1 protein levels in the hypothalamus after control and knockdown AAV injection. β-Actin served as the loading control ( n = 5 mice). rAAV-saCas9-sgRNA1, rAAV-saCas9-sgRNA2, and rAAV-saCas9-sgControl (recombinant adeno-associated viruses encoding sgRNA1, sgRNA2, and a non-targeting control sgRNA, respectively) were used. E: A schematic diagram of bilateral injection of Cre-dependent AAV-DIO-saCas9-sgRNAs and its control into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. F: Representative IF staining of the HA-Tag signifying the expression of saCas9 in AgRP neurons ( n = 3 mice; scale bars, 100 μm). Data are presented as the mean ± standard error of the mean. *** P < 0.001 by unpaired t -tests (D). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; DIO, double-floxed inverted open reading frame; NPY, neuropeptide Y; RAF1, v-raf-leukemia viral oncogene 1; sgRNA, single guide RNA.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: In Vivo, Biomarker Discovery, Nucleic Acid Electrophoresis, Western Blot, Control, Knockdown, Injection, Recombinant, Staining, Expressing

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 knockout in AgRP neurons in mice did not alter their metabolic phenotypes under NCD feeding. A: Representative image of control and AgRP- Raf1 -KO mice fed an NCD. B–J: Various metabolic indicators including body weight gain curves ( n = 8 mice; B), food intake ( n = 8 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 3–4 mice; G), energy expenditure ( n = 3–4; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. ns P > 0.05 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; KO, knockout; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Knock-Out, Control, Staining

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 knockout in AgRP neurons protected against DIO. A: Representative image of control and AgRP- Raf1 -KO mice fed an HFD. B–J: Various metabolic indicators, including body weight gain curves ( n = 8–10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6–7 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t-tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; KO, knockout; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Knock-Out, Control, Staining

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 regulated MAPK signaling under insulin stimulation. A: Relative mRNA levels of Raf1 , Agrp , and Npy in the hypothalamus of control and AgRP- Raf1 -OE mice fed an NCD ( n = 5–6 mice). B: Western blotting analysis of protein levels of FLAG, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, CREB, and pCREB in the N42 Raf1 -overexpression cells. β-Actin served as the internal control ( n = 3). C: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -overexpression cells. D: Western blotting analysis of protein levels of RAF1, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, and pCREB in the N42 Raf1 -knockout cells. β-Actin served as the internal control ( n = 3). E: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -knockout cells. F: Representative IF staining of pCREB in AgRP neurons of control and AgRP- Raf1 -OE mice following 2 mU insulin (icv) stimulation ( n = 3 mice; scale bars, 100 μm). G: Fluorescence intensity quantification of pCREB co-localized with HA/mCherry ( n = 3 mice; AAV-DIO-mCherry, N = 77; AAV-DIO- Raf1 -HA, N = 105). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (A, C, E, and G). Abbreviations: AgRP, agouti-related peptide; CREB, cAMP response element-binding protein; ERK1/2, extracellular signal-regulated kinases 1 and 2; icv, intra-cerebroventricular injection; IF, immunofluorescence; MEK1/2, mitogen-activated protein kinase kinases 1 and 2; NCD, normal chow diet; NPY, neuropeptide Y; OE, overexpression; p-CREB, phospho-CREB; p-ERK1/2, phospho-ERK1/2; p-MEK1/2, phospho-MEK1/2; RAF1, v-raf-leukemia viral oncogene 1.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Control, Western Blot, Over Expression, Phospho-proteomics, Knock-Out, Staining, Fluorescence, Binding Assay, Injection, Immunofluorescence

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Role of hypothalamic AgRP neuron Raf1 in energy homeostasis regulation. This graphic abstract illustrates the pivotal role of Raf1 within hypothalamic AgRP neurons in governing energy homeostasis. Specifically, Raf1 exerts its regulatory function via the MAPK-mediated modulation of Agrp and Npy expression. In normal physiological conditions, this mechanism contributes to the maintenance of energy balance. Under HFD challenges, the RAF1-MAPK-AGRP/NPY axis becomes dysregulated, leading to disruptions in energy homeostasis. Abbreviations: DIO, diet-induced obesity; HFD, high-fat diet; KO, knockout; OE, overexpression.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Expressing, Knock-Out, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: Sorafenib (an RAF1 inhibitor) significantly attenuated STOML2-induced cell cycle progression, migration and autophagy, while promoting apoptosis in HCC cells. (A) Cell cycle distribution analysis of Huh7-STOML2 and control cells following treatment with sorafenib (20 μ M, 24 h) or DMSO. Representative images from three independent experiments are shown. (B) Wound healing assay showing the migratory capacity of Huh7-STOML2 cells treated with sorafenib or DMSO. Representative images from three independent experiments are shown. (C) Flow cytometric analysis of apoptosis in STOML2-overexpressing Huh7 and control cells after treatment with sorafenib or DMSO (unpaired Student's t-test, **** P<0.0001). Representative flow cytometry plots and quantitative data (mean ± SD) from three independent experiments are shown. (D) Transwell assay showing the invasive capacity of Huh7-STOML2 and control cells following sorafenib treatment or DMSO. Data are presented as mean ± SD (unpaired Student's t-test, *** P<0.001). Western blot analysis of (E) MAPK signaling pathway proteins, and (F) p62 and Beclin1 in Huh7-STOML2 cells after treatment with sorafenib or DMSO. Representative images from three independent experiments are shown. p-, phosphorylated; STOML2, stomatin-like protein 2.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

Techniques: Migration, Control, Wound Healing Assay, Flow Cytometry, Transwell Assay, Western Blot

Journal: Poultry Science

Article Title: Curcumin alleviates proliferation dysfunction in chicken granulosa cells under oxidative stress through AKT-Raf1-ERK1/2 signaling pathway

doi: 10.1016/j.psj.2025.105342

Figure Lengend Snippet: Curcumin modulates the AKT-Raf1-ERK1/2 signaling pathway in chicken granulosa cells under oxidative stress. (A) Protein expression of AKT1, Raf1, and ERK1/2 under oxidative stress and curcumin treatment. (B) Relative expression of AKT. (C) Relative expression of p-AKT. (D-E) Changes in the relative expression and phosphorylation levels of Raf1. (F-G) Changes in the relative expression and phosphorylation levels of ERK1/2. The results are presented in the form of mean ± SD. n = 4. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns p ≥ 0.05.

Article Snippet: ZenBio , Phospho-Raf1 (Ser259) Rabbit pAb , 1: 1000.

Techniques: Expressing, Phospho-proteomics

Journal: Poultry Science

Article Title: Curcumin alleviates proliferation dysfunction in chicken granulosa cells under oxidative stress through AKT-Raf1-ERK1/2 signaling pathway

doi: 10.1016/j.psj.2025.105342

Figure Lengend Snippet: Curcumin modulates the AKT-Raf1-ERK1/2 signaling pathway in chicken granulosa cells under oxidative stress. (A) Protein expression of AKT1, Raf1, and ERK1/2 under oxidative stress and curcumin treatment. (B) Relative expression of AKT. (C) Relative expression of p-AKT. (D-E) Changes in the relative expression and phosphorylation levels of Raf1. (F-G) Changes in the relative expression and phosphorylation levels of ERK1/2. The results are presented in the form of mean ± SD. n = 4. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns p ≥ 0.05.

Article Snippet: ZenBio , Raf1 Rabbit pAb , 1: 1000.

Techniques: Expressing, Phospho-proteomics

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot confirming effective silencing of ARAF, BRAF, or CRAF expression in iM27 cells using corresponding siRNAs. (B) Representative fluorescence microscopy images showing β-catenin expression in iM27 cells transfected with scramble siRNA or siRNAs that deplete ARAF, BRAF, or CRAF expression under PLX4032 treatment. iM27 transfected with scramble siRNA without PLX4032 treatment were used as a control. (C) Quantification of nuclear β-catenin intensity in iM27 and genetically modified iM27 cells as shown in (B) with or without PLX4032 treatment using ImageJ. The data are presented as the means ± SDs (n = 11 randomly selected 20 X fields per group). (D) Representative PLA images showing BRAF-CRAF heterodimerization in DMSO- and PLX4032-treated iM27 cells. Red dots indicate BRAF-CRAF dimers. (E) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (F) Representative PLA images showing BRAF-BRAF homodimerization in DMSO- and PLX4032-treated iM27 cells. The red dots indicate BRAF-BRAF dimers. (G) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (H) Representative PLA images showing CRAF-CRAF homodimerization in DMSO- and PLX4032-treated iM27 cells. The red dots indicate CRAF-CRAF dimers. (I) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (J) Western blot analysis showing increased CRAF-CRAF and CRAF-BRAF interactions in PLX4032-treated iM27 cells compared with those in DMSO-treated iM27 cells. Co-IP was performed via the use of an anti-Myc antibody to pull down interacting proteins in iM27 cells coexpressing Myc-tagged and Flag-tagged CRAF. (K-M) Western blot analysis of phosphorylated BRAF (Ser445) and CRAF (Ser338) expression in iM27 (K), BRAF-deficient iM27 overexpressing wild-type BRAF or BRAF (T529N) (L), and CRAF-deficient iM27 overexpressing wild-type CRAF or CRAF (T421N) (M) treated with either DMSO or PLX4032.

Article Snippet: Afterwards, the cells were blocked with Duolink blocking solution for one hour at 37°C and then incubated overnight at 4°C with the following primary antibodies: BRAF (Proteintech, 20899-1-AP), CRAF (Proteintech, clone 1D6A1), FLAG (Proteintech, 20543-1-AP), or MYC (Proteintech, clone 1A5A2).

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Transfection, Control, Genetically Modified, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot confirming effective silencing of KRAS, HRAS, and NRAS expression in iM27 using siRNAs. (B) PLA results showing BRAF and CRAF heterodimerization in iM27 transfected with scramble siRNA (Scr), scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. (C) Western blot showing the phosphorylation of BRAF (Ser445) and CRAF (Ser338) in iM27 transfected with scramble siRNA and RAS-deficient iM27 with and without PLX4032 treatment. (D) Confocal images of nuclei visualized by Hoechst staining in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. The nuclear boundaries are outlined with yellow circles. (E-F) Quantification of nuclear morphology based on the confocal images shown in (D), including the nuclear aspect ratio (E) and circularity (F), was performed via ImageJ. The data are presented as the means ± SDs (n = 30-67 nuclei per group). (G) Confocal images of F-actin expression and organization in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032 (scramble), and RAS-deficient iM27 (siRASs) treated with PLX4032. Insets display enlarged views of representative individual cells (highlighted by yellow boxes). Yellow arrows indicate actin caps. (H-I) Quantification of F-actin intensity (H) and actin cap intensity (I) using ImageJ. The data are presented as the means ± SDs (n = 6 randomly selected 20 X fields per group for H; n = 25-52 nuclei per group for I). (J) Confocal images showing Nesprin-2 distribution in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. The yellow arrow indicates abnormal cytosolic localization of Nesprin-2. (K) Representative fluorescence images showing nuclear β-catenin staining in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. (L) Quantification of nuclear β-catenin intensity under the indicated conditions shown in (K). The data are presented as the means ± SDs (n = 9 randomly selected 20X fields per group).

Article Snippet: Afterwards, the cells were blocked with Duolink blocking solution for one hour at 37°C and then incubated overnight at 4°C with the following primary antibodies: BRAF (Proteintech, 20899-1-AP), CRAF (Proteintech, clone 1D6A1), FLAG (Proteintech, 20543-1-AP), or MYC (Proteintech, clone 1A5A2).

Techniques: Western Blot, Expressing, Transfection, Phospho-proteomics, Staining, Fluorescence

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot showing increased ERK phosphorylation in response to increasing concentrations of PLX4032 in iM27 cells. (B) Western blot showing the phosphorylation of ERK in iM27 transfected with scramble siRNA and RAS-deficient iM27 (siRASs) with and without PLX4032 treatment. (C-D) Western blots showing ERK phosphorylation in BRAF-deficient iM27 overexpressing wild-type BRAF and BRAF(T529N) (C) and in CRAF-deficient iM27 overexpressing wild-type CRAF and CRAF(T421N) (D) with and without PLX4032 treatment. (E) Western blot showing increased phosphorylation of MYPT1 in PLX4032-treated iM27. (F) Western blot showing the phosphorylation of MYPT1 in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ERK inhibitor SCH772984 (SCH). (G) Representative confocal images of nuclear morphology visualized by Hoechst staining in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. (H-I) Quantification of nuclear morphology based on the confocal images shown in (G), including the nuclear aspect ratio (H) and circularity (I), was performed via ImageJ. The data are presented as the means ± SDs (n = 27-38 nuclei per group). (J) Confocal images of F-actin expression and organization in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. Insets display enlarged views of representative individual cells (highlighted by yellow boxes). Yellow arrows indicate the actin caps. (K) Quantification of the actin cap intensity via ImageJ shown in (J). The data are presented as the means ± SDs (n = 20 nuclei per group). (L) Confocal images showing SUN2 distribution in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. The yellow arrow indicates abnormal nuclear localization of SUN2 in PLX4032-treated cells. (M) Representative fluorescence images showing nuclear β-catenin staining in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. (N) Quantification of the nuclear β-catenin intensity under the indicated conditions shown in (M). The data are presented as the means ± SDs (n = 40 nuclei per group).

Article Snippet: Afterwards, the cells were blocked with Duolink blocking solution for one hour at 37°C and then incubated overnight at 4°C with the following primary antibodies: BRAF (Proteintech, 20899-1-AP), CRAF (Proteintech, clone 1D6A1), FLAG (Proteintech, 20543-1-AP), or MYC (Proteintech, clone 1A5A2).

Techniques: Western Blot, Phospho-proteomics, Transfection, Staining, Expressing, Fluorescence