atcc 29257  (ATCC)

Journal: Frontiers in Pharmacology

Article Title: Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs

doi: 10.3389/fphar.2025.1572878

Figure Lengend Snippet: Apigenin promotes pigmentation independent of the classic MC1R/cAMP/PKA-pigmentation pathway in HEMCs. (A) HEMCs were treated with apigenin (10 μM) for the indicated time periods (0–180 min), and the phosphorylation of PKA and CREB was assessed by Western blotting. (B) HEMCs were pretreated with or without 10 μM DDA (an AC inhibitor) for 1 h before the addition of apigenin for 48 h. Melanin content, as well as the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a, were measured as described previously. (C) HEMCs were pretreated with or without 10 μM N-1A (an MC1R inhibitor) for 1 h before apigenin treatment for 48 h. Melanin content and the expression levels of Tyrosinase, Cdc42, and Rab27a were measured as described previously. Data are expressed as the mean ± SEM (n = 3). *p < 0.05 vs. untreated cells. MC1R, melanocortin-1 receptor; cAMP, Cyclic Adenosine Monophosphate; PKA, protein kinase A; HEMCs, human epidermal melanocytes; CREB, cAMP response element-binding protein; AC, adenylate cyclase; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a.

Article Snippet: Anti-Myosin Va (sc-365986, 1:200), KIF5b (sc-133184, 1:200), Cdc42 (sc-8401, 1:200), Rab27a (sc-74586, 1:200), p-c-KIT (sc-365504, 1:200), c-KIT (sc-13508, 1:200), p-Raf-1 (sc-271929, 1:200), Raf-1 (sc-7267, 1:200), p-p38 (sc-166182, 1:200), p38 (sc-398546, 1:200), p-ERK (sc-7383, 1:200), ERK (sc-514302, 1:200), p-RSK (sc-377526, 1:200), RSK (sc-74575, 1:200), p-MEK (sc-81503, 1:200), MEK (sc-13159, 1:200), MSK1 (sc-518173, 1:200), p-PKA (sc-293036, 1:200), PKA (sc-390548, 1:200), p-CREB (sc-81486, 1:200), CREB (sc-240, 1:200), CRTC1 (sc-271333, 1:200), c-KIT siRNA (sc-29225), Rab 27a siRNA (sc-41834) and Cdc42 siRNA (sc-29256), siRNA Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) were obtained from Santa Cruz Biotechnology (Dallas, United States).

Techniques: Phospho-proteomics, Western Blot, Expressing, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs

doi: 10.3389/fphar.2025.1572878

Figure Lengend Snippet: Apigenin promotes pigmentation through the c-KIT-CRTCs/CREB signaling pathway. (A,B) HEMCs were pretreated with or without 10 μM ISCK03 (c-KIT inhibitor) for 1 h before apigenin was added for an additional 1 h. Phosphorylated CREB (p-CREB) and nuclear levels of CRTC1 were measured by Western blotting. Histone H3 served as a reference for nuclear proteins, and β-actin served as a reference for cytosolic proteins. (C,D) HEMCs were pretreated with or without 10 μM ISCK03 for 1 h before apigenin was added for 48 h. Melanin content and the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a were measured as described previously. (E,F) HEMCs were transfected with si-NC or si-c-KIT for 24 h, followed by apigenin (10 μM) treatment for 48 h. Melanin content and the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a were measured as described previously. (G) Binding of c-KIT with apigenin was analyzed by microscale thermophoresis (MST). The binding curve represents data points from 3 measurements. The calculated Kd is 2.6 ± 0.14 µM. Data are expressed as means ± SEM (n = 3). *p < 0.05 vs. non-treated cells, # p < 0.05 vs. apigenin-treated cells. c-KIT, cellular-KIT; CREB, cAMP response element-binding protein; CRTC, CREB-regulated co-activator; HEMCs, human epidermal melanocytes; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a.

Article Snippet: Anti-Myosin Va (sc-365986, 1:200), KIF5b (sc-133184, 1:200), Cdc42 (sc-8401, 1:200), Rab27a (sc-74586, 1:200), p-c-KIT (sc-365504, 1:200), c-KIT (sc-13508, 1:200), p-Raf-1 (sc-271929, 1:200), Raf-1 (sc-7267, 1:200), p-p38 (sc-166182, 1:200), p38 (sc-398546, 1:200), p-ERK (sc-7383, 1:200), ERK (sc-514302, 1:200), p-RSK (sc-377526, 1:200), RSK (sc-74575, 1:200), p-MEK (sc-81503, 1:200), MEK (sc-13159, 1:200), MSK1 (sc-518173, 1:200), p-PKA (sc-293036, 1:200), PKA (sc-390548, 1:200), p-CREB (sc-81486, 1:200), CREB (sc-240, 1:200), CRTC1 (sc-271333, 1:200), c-KIT siRNA (sc-29225), Rab 27a siRNA (sc-41834) and Cdc42 siRNA (sc-29256), siRNA Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) were obtained from Santa Cruz Biotechnology (Dallas, United States).

Techniques: Western Blot, Expressing, Transfection, Binding Assay, Microscale Thermophoresis

Journal: Frontiers in Pharmacology

Article Title: Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs

doi: 10.3389/fphar.2025.1572878

Figure Lengend Snippet: A proposed model showing that apigenin promotes pigmentation through the c-KIT/Raf-1/MAPK/CREB signaling pathway. Apigenin activates the c-KIT receptor, leading to phosphorylation of Raf-1. Once activated, Raf-1 phosphorylates and activates MAPK, resulting in CREB phosphorylation and subsequent nuclear translocation of CRTCs. Phosphorylated CREB, in association with CRTCs, promotes MITF transcription, which in turn induces the expression of Tyrosinase, Rab27a, and Cdc42. This cascade of events drives melanosome maturation and transport. Tyrosinase, TRP-1, and TRP-2 are key regulators of melanosome maturation. Cdc42 facilitates dendrite extension and filopodia formation, while Rab27a interacts with its effectors, Mlph and Myosin Va, to regulate actin-dependent melanosome transport and anchoring to the plasma membrane. c-KIT, cellular-KIT; Raf-1, rapidly accelerated fibrosarcoma-1; MAPK, mitogen-activated protein kinase; CRTC, CREB-regulated co-activator; CREB, cAMP response element-binding protein; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a; TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2.

Article Snippet: Anti-Myosin Va (sc-365986, 1:200), KIF5b (sc-133184, 1:200), Cdc42 (sc-8401, 1:200), Rab27a (sc-74586, 1:200), p-c-KIT (sc-365504, 1:200), c-KIT (sc-13508, 1:200), p-Raf-1 (sc-271929, 1:200), Raf-1 (sc-7267, 1:200), p-p38 (sc-166182, 1:200), p38 (sc-398546, 1:200), p-ERK (sc-7383, 1:200), ERK (sc-514302, 1:200), p-RSK (sc-377526, 1:200), RSK (sc-74575, 1:200), p-MEK (sc-81503, 1:200), MEK (sc-13159, 1:200), MSK1 (sc-518173, 1:200), p-PKA (sc-293036, 1:200), PKA (sc-390548, 1:200), p-CREB (sc-81486, 1:200), CREB (sc-240, 1:200), CRTC1 (sc-271333, 1:200), c-KIT siRNA (sc-29225), Rab 27a siRNA (sc-41834) and Cdc42 siRNA (sc-29256), siRNA Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) were obtained from Santa Cruz Biotechnology (Dallas, United States).

Techniques: Phospho-proteomics, Translocation Assay, Expressing, Clinical Proteomics, Membrane, Binding Assay

Journal: Molecular Cancer

Article Title: A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

doi: 10.1186/s12943-022-01494-z

Figure Lengend Snippet: miR-10b regulates alternative splicing of CDC42, thereby controlling its total levels via U6 regulation. a Alternative splicing events induced by miR-10b inhibitor and U6 ASO #1, as determined by RNAseq ( n = 3). Venn diagrams indicate the numbers of exon skipping (SE) and alternative 3′ splice sites (A3SS) modulation, in the indicated conditions. b Schematic illustration of two major CDC42 mRNA isoforms, with alternative exons encoding alternative 5′ and 3′ UTRs (left panel). Expression analysis of CDC42 isoforms in the normal brain ( n = 1141), low grade glioma (LGG) ( n = 509) and GBM ( n = 153) in TCGA database demonstrates that ENST00000344548 (CDC42-iso2) is the pathologic variant associated with glioma progression, whereas ENST00000315554 (CDC42-iso1) is present at similarly low levels in the normal brain, LGG, and GBM (right panel). c qRT-PCR analysis of CDC42-iso1 or iso2 mRNAs in glioma cells and GSCs transfected with either U6 ASOs, or miR-10b inhibitor or mimic. The results are expressed as the fold-changes relative to the corresponding control groups (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. d Western blot analysis of the indicated CDC42 forms in the cells transfected with either U6 ASO, or miR-10b inhibitor or mimic. e Western blot analysis of the cells transfected with either selective siRNA targeting iso2 (siCDC42-iso2) or both CDC42 isoforms (siCDC42-total) demonstrates that KD of iso2 is sufficient for reducing total CDC42 levels. f Analysis of GSC spheroids transfected with either siCDC42-iso2 or siCDC42-total demonstrates that CDC42 KD reduces GSC growth. The number and size of GSC spheres have been monitored using Image J at day 7 after transfection (mean ± SD, n = 3, 8 images per cultured analyzed). P values were calculated using two-tail unpaired t-test. g Analysis of glioma LN229 cells transfected with either siCDC42-iso2 or siCDC42-total demonstrates that CDC42 KD reduces glioma growth. Cell viability was monitored 72 h after transfection by WST-1 assay (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. h Representative images of LN229 cells immuno-stained for Ki-67 (green) and DAPI staining (blue) and quantification of the Ki67 + cells (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. i Schematic illustration of alternative splicing of CDC42, regulated by miR-10b via its binding to U6 snRNA. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance

Article Snippet: CDC42-total siRNA was a pool of 4 siRNAs targeting both isoforms (CUCCUGAUAUCCUACACAA; CCUAGAUCUAGUUUAGAAA; GAGUAGAUCCAGUAUUUGA; CCCAAACCUAAUUCUUGUA), purchased from Santa Cruz Biotechnology (sc-29,256).

Techniques: Alternative Splicing, Expressing, Variant Assay, Quantitative RT-PCR, Transfection, Control, Western Blot, Cell Culture, WST-1 Assay, Staining, Binding Assay