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: Theranostics

Article Title: Targeting CAMK1D-engineered nanoactivator suppresses cancer stem cell maintenance and immune evasion in enzalutamide-resistant prostate cancer

doi: 10.7150/thno.120826

Figure Lengend Snippet: EC@HNA effectively reverses ENZR in vivo . (A) Schematic representation of the in vivo experimental procedure. Orthotopic prostate tumors were established by injecting 1 × 10⁵ luciferase-labeled RM-1 cells per mouse. After tumor fluorescence was detected, mice were randomly assigned to eight groups and received various formulations of siCAMK1D (3 μg/mouse) or ENZ (5 mg/kg) via tail vein injection every three days. (B) Bioluminescent images showing tumor growth in mice subjected to different treatments using Living Image software. (C) Quantification of bioluminescence signals from tumor-bearing mice after various treatments (n=5). (D) Survival rates of subcutaneous RM-1 tumor-bearing mice treated with the indicated therapies (n = 10). (E) Western blot analysis of stemness markers and CAMK1D expression in PCa xenografts. (F) Immunofluorescence staining of CAMK1D and phosphorylated CREB in mouse tumor tissues. (G) Immunofluorescence images depicting Ki67, CD44, and TUNEL staining in PCa tissue sections. The areas outlined with black borders indicate normal prostatic glandular tissue, whereas the remaining regions correspond to tumor tissue. Data were assessed for normal distribution using the Shapiro-Wilk test, and statistical comparisons were performed using two-sided Student's t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For immunofluorescence analysis, 4 μm tissue sections were fixed and incubated to staining with primary antibodies against CAMK1D (1:100, Abcam, catalog no. ab198165), CD44 (1:100, Proteintech, catalog no. 15675-1-AP), CD133 (1:100, Proteintech, catalog no. 18470-1-AP), CREB (1:100, Proteintech, catalog no. 12208-1-AP; 1:100, Abcam, catalog no. ab178322), and P63 (1:600, Abcam, catalog no. ab124762).

Techniques: In Vivo, Luciferase, Labeling, Fluorescence, Injection, Software, Western Blot, Expressing, Immunofluorescence, Staining, TUNEL Assay