Journal: JID Innovations

Article Title: Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

doi: 10.1016/j.xjidi.2025.100444

Figure Lengend Snippet: The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of Notch 1 (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Article Snippet: Membranes were blocked with 10% nonfat dry milk for 1 hour and incubated with primary antibodies against NOTCH 1 (number 3608, Cell Signaling Technology), cleaved NOTCH-1 (number 4147, Cell Signaling Technology), c-MYC, or β-actin (ab8227, Abcam) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Fluorescence, Labeling, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Control, High Content Screening

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 1 A Notch signature is present in MBMs. A-B GSE200218: (A) Uniform Manifold Approximation and Projection (UMAP) showing the combined tumor cell cluster of melanoma brain (n = 15) and peripheral (n = 10) metastases (top), with Notch1 signature distribution among the two (bottom). B Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) in brain tumor cell cluster compared to the peripheral metastases. C Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) in GSE200217 in MBM compared to extracranial metastases. D Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) for GSE50496

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 2 Notch1 blockade affects histone expression. A Bubble plot depicting the most significantly inhibited pathways in K457 melanoma cells treated with 50ug/ml anti-Notch1 for 48 h. B Heat Map depicting histone mRNAs significantly downregulated in the cells from A. C-D Core histone protein levels in K457 (C) and A375 (D) cells treated as in A. α-tubulin was used as loading control. Band intensity is the mean between two experimental repeats for each cell line, normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 treated cells normalized to ctrl

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques: Expressing, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 3 Notch1 blockade causes replication stress and DNA damage. A % of K457 cells in each phase of the cell cycle at 48 h treatment with anti-N1 (100ug/ml). B DNA replication fork speed in K457 cells. 100 fibers were analyzed for each group. Speed: IdU (red) tract length/time of the IdU pulse (30 min) = um/min. um were converted to Kilobases (Kb) using the conversion factor 1um = 2.59 kb. C pChK1S345 is induced because of RF stress and ATR activation. Band intensity is the mean between two experimental repeats normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 treated cells normalized to ctrl. D γH2 AX foci in K457 cells treated as in A. n ≥ 50 nuclei per section were counted for 5 section per group. E Neutral Comet Assay of K457 cells treated as in A. n ≥ 200 comets/group. Significance of the differences among groups was determined by the Student’s t test. Data are the mean of three independent experiments each performed in triplicate

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques: Activation Assay, Control, Neutral Comet Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 4 Concurrent blockade of Notch1 and ChK1 increases replication stress and DNA damage. A pChK1S345, pChK1S296 (autophosphorylation site that leads to full activity) and total ChK1 in K457 cells treated with anti-N1 (100ug/ml) and prexasertib (prex) (5 nM), alone or in combination, for 48 h. Band intensity is the mean between two experimental repeats normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 and prex treated cells normalized to ctrl. B DNA replication fork speed. 100 fibers were analyzed for each group. Speed: IdU (red) tract length/time of the IdU pulse (30 min) = um/min. um were converted to Kilobases (Kb) using the conversion factor 1um = 2.59 kb.). Representative DNA tracts are shown on the right. C % of K457 cells treated as in A, in each phase of the cell cycle. Significance between treatment for each phase was calculated by the Student’s t test (table below). D γH2 AX foci in cells treated as in A. n ≥ 50 nuclei per section, 5 section per group. Representative pictures are shown below. E Neutral Comet Assay of cells treated as in A. n ≥ 200 comets/group. Representative comets are shown below. Data are the mean of three independent experiments each performed in triplicate. Significance of the differences among groups was determined by the Student’s t test

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques: Activity Assay, Control, Neutral Comet Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 5 Concurrent blockade of Notch1 and ChK1 causes mitotic catastrophe and cell death. A % of A375 cells positive for cleaved-caspase-3 (apoptotic), KI67 (proliferative) or double positive (mitotic catastrophe) after treatment with anti-N1 (100ug/ml) and prexasertib (5 nM), alone or in combination, for 24 h. Representative staining of each condition is shown on the right (scale bar: 10um). B Survival of A375 at 48 h post treatment determined by trypan blue staining. C Clonogenic assay of A375 treated with anti-N1, prex and a combination of the two. A picture of representative colonies for each condition is shown on the right. D normal human melanocytes and human fibroblasts treated as the cells in B. Statistical differences were determined by the Student’s t test. Data are the mean of three independent experiments each performed in triplicate

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques: Staining, Clonogenic Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 6 Concurrent blockade of Notch1 and ChK1 improves the survival of animals bearing MBMs. A 105 A375 cells expressing luciferase, were inoculated stereotactically into the right cerebral cortex of athymic nude mice. Tumors were monitored twice weekly since inoculation. Kaplan–Meier curves were generated with Graph Pad Prism10. Logrank p values, median overall survival (OS) and Hazard Ratio (HR) are shown. B Representative H&E staining. Scale bar: 200um

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques: Expressing, Luciferase, Generated, Staining