normal human epidermal melanocytes nhem  (PromoCell)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70

doi: 10.1007/s00018-025-05939-8

Figure Lengend Snippet: G3BP1 and (phospho-)EIF2S1 expression in malignant melanoma. ( a ) Western blot analysis of G3BP1 protein expression in normal human epidermal melanocytes (NHEM) from different donors and passages (P5–P14), and in melanoma cell lines derived from metastases (SK-Mel-28, SK-Mel-3) and primary tumors (WM1366, Mel Juso, Mel Ho, WM35). ACTINB served as a loading control. Densitometric quantification of G3BP1 level is shown. ( b ) Analysis of G3BP1 expression in skin cutaneous melanoma (SKCM) samples (red) compared to non-tumor samples (grey). Data obtained from GEPIA database, p-value cut-off = 0.01, |Log2FC| Cut-off = 1, retrieved on August 21, 2025. (c) Immunofluorescence staining of G3BP1 (green) in tissue sections of malignant melanoma, illustrating representative samples with positive (+) and negative (–) G3BP1 staining. DAPI (blue) was used for nuclear counterstaining. Hematoxylin and eosin (HE) staining was performed to visualize tissue morphology. Quantification of G3BP1-positive and -negative samples in tissue microarrays (nevi: n = 12; melanoma: n = 13) is shown. ( d ) Western blot analysis of phosphorylated (P-) and total EIF2S1 in melanoma and non-melanoma cell lines following sodium arsenite treatment (+ SA) (600 µM) or under untreated conditions. (e ) Quantification of the P-EIF2S1/EIF2S1 ratio in each cell line (mean of n = 3 biological replicates). ( f ) Immunofluorescence staining of G3BP1 in Mel Im melanoma cells following treatment with 600 µM sodium arsenite (SA), 2,2′-Dipyridyl (DP), 2-(N-morpholino)ethanesulfonic acid (MES), or hydrogen peroxide (H₂O₂), to assess stress-induced SG formation. Magnification: 40x. Data are presented as mean ± SEM (** p < 0.01)

Article Snippet: Normal Human Epidermal Melanocytes derived from neonatal foreskin (NHEM, PromoCell, Heidelberg, Germany) were cultivated in M2 melanocyte growth medium (M2 with Supplementary Mix containing growth factors and hormones; PromoCell, Heidelberg, Germany) under a humidified atmosphere of 5% CO 2 at 37 °C.

Techniques: Expressing, Western Blot, Derivative Assay, Control, Immunofluorescence, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70

doi: 10.1007/s00018-025-05939-8

Figure Lengend Snippet: Stress granule (SG) formation in response to hypoxic, oxidative, and osmotic stress. (a) Immunofluorescence staining of G3BP1 in melanoma cell lines Mel Juso and Mel Im following treatment with 600 µM sodium arsenite (SA) or Desferrioxamine (DFX), an iron chelator that mimics hypoxia. SG formation is indicated by punctate G3BP1-positive structures. (b) G3BP1 immunofluorescence staining of Mel Juso cells incubated under hypoxic (1% O₂) or normoxic (21% O₂) conditions for 24 h. SG formation was quantified as the percentage of G3BP1-positive cells. Data represent the mean ± SEM from three independent experiments (unpaired Student’s t-test; ns = not significant). (c) Immunofluorescence staining of G3BP1 in normal human epidermal melanocytes (NHEM) treated with or without 600 µM sodium arsenite, under control conditions or following exposure to 80 mJ/cm² UVC irradiation (image sections *, #: 40x magnification)

Article Snippet: Normal Human Epidermal Melanocytes derived from neonatal foreskin (NHEM, PromoCell, Heidelberg, Germany) were cultivated in M2 melanocyte growth medium (M2 with Supplementary Mix containing growth factors and hormones; PromoCell, Heidelberg, Germany) under a humidified atmosphere of 5% CO 2 at 37 °C.

Techniques: Immunofluorescence, Staining, Incubation, Control, Irradiation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70

doi: 10.1007/s00018-025-05939-8

Figure Lengend Snippet: Melanoma cells exhibit high levels of HSP70 and p-G3BP1. (a) Western blot analysis of phosphorylated G3BP1 (P-G3BP1, Ser149) in normal human epidermal melanocytes (NHEM; passages P5 and P6) and melanoma cell lines. Additional tumor cell lines including Panc-1 (pancreatic carcinoma), Hep3B and HepG2 (hepatocellular carcinoma), and HT29 and SW480 (colorectal carcinoma) were included for comparison. ACTINB served as a loading control. (b) Densitometric quantification of P-G3BP1/G3BP1 protein ratio in NHEM, SK-Mel-28, WM1366, Mel Ho, Mel Juso, Sk-Mel-3 and WM35 cells. NHEM protein expression was set to 1. Normalized to ACTINB. (c) RNA sequencing analysis of HSP70 paralogues (HSPA1A, HSPA1B) in melanoma cell lines and NHEM. Read counts were normalized to library size. (d) Quantitative real-time PCR of HSPA1A and HSPA1B mRNA levels in melanoma cell lines and NHEM. (e) Western blot analysis of total HSP70 protein expression in NHEM (passages P5 and P8) and melanoma cell lines. ACTINB served as a loading control. (f) Western blot analysis of p-G3BP1 expression following treatment of Mel Juso and Mel Im cells with hypoxia-mimetic agents Desferrioxamine (DFX) and Dimethyloxalylglycine (DMOG). ACTINB was used as a loading control. (g) Western blot analysis of P-G3BP1 and G3BP1 expression in Mel Juso cells exposed to hypoxic conditions (1% O₂). ACTINB served as loading controls. (h) Western blot analysis of HSP70 expression in Mel Juso and Mel Im cells treated with DFX and DMOG. ACTINB served as a loading control. (i) Western blot analysis of HSP70 expression in Mel Juso cells under hypoxic conditions (1% O₂). ACTINB was used as a loading control

Article Snippet: Normal Human Epidermal Melanocytes derived from neonatal foreskin (NHEM, PromoCell, Heidelberg, Germany) were cultivated in M2 melanocyte growth medium (M2 with Supplementary Mix containing growth factors and hormones; PromoCell, Heidelberg, Germany) under a humidified atmosphere of 5% CO 2 at 37 °C.

Techniques: Western Blot, Comparison, Control, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70

doi: 10.1007/s00018-025-05939-8

Figure Lengend Snippet: Caseinkinase 2a regulates Stress Granule Formation by modulating G3BP1 phosphorylation (a) Analysis of CSNK2A1 expression in skin cutaneous melanoma (SKCM; T) samples (red) compared to non-tumor skin (N) samples (grey). Data were obtained from the GEPIA database with a p-value cut-off of 0.01 and |Log₂FC| cut-off of 1 (retrieved August 21, 2025). (b) Representative western blot analysis of casein kinase 2α (CK2α) expression in normal human epidermal melanocytes (NHEMs) from different donors and melanoma cell lines (SK-MEL-28, WM1366, SK-MEL-3, Mel Juso, Mel Ho, and WM35). ACTINB was used as a loading control. (c) Schematic representation of the experimental timeline for TBCA treatment. (d) Immunofluorescence staining of G3BP1 (red) in Mel Juso cells treated with TBCA or DMSO for 45 min (stress phase) or after 24 h (recovery phase). Nuclei were counterstained with DAPI (blue). Quantification of stress granule (SG)-positive cells per field of view is shown as the mean of three independent experiments (lower panels). (e) Representative western blot analysis of HSP70, phosphorylated G3BP1 (P-G3BP1), and CK2α in cells treated with TBCA or DMSO, followed by sodium arsenite (SA) treatment for 45 min (stress phase) or 24 h (recovery phase). ACTINB served as a loading control

Article Snippet: Normal Human Epidermal Melanocytes derived from neonatal foreskin (NHEM, PromoCell, Heidelberg, Germany) were cultivated in M2 melanocyte growth medium (M2 with Supplementary Mix containing growth factors and hormones; PromoCell, Heidelberg, Germany) under a humidified atmosphere of 5% CO 2 at 37 °C.

Techniques: Phospho-proteomics, Expressing, Western Blot, Control, Immunofluorescence, Staining