Journal: The Journal of cell biology

Article Title: Aberrant phosphorylation inactivates Numb in breast cancer causing expansion of the stem cell pool.

doi: 10.1083/jcb.202112001

Figure Lengend Snippet: Figure 3. Characterization of Numb phosphomutants. (A) Scheme showing the behavior of a SC (A) and daughter progenitor cell (B) in WT vs. Numb-KO (Tosoni et al., 2015). (B) Scheme of the growth of MS from WT and Numb-KO MECs (Tosoni et al., 2015). (C and D) WT and Numb-KO cells, transduced with the indicated constructs (DsRed fusion proteins; EV, empty vector), were assessed for SFE (C, by counting only red cells or MS) and size (D, N = number of epifluorescent MS analyzed). Results are expressed relative to WT cells (see also Table S2). Significance was calculated vs. EV cells. Representative images of the MS are in D, top panel. Bar, 100 µm. (E) WT and Numb-KO MS, transduced with the indicated constructs (Flag-tagged), were analyzed by IB. Arrows, endogenous (black) or overexpressed (red) Numb (also in G). Activated Notch (Act. Notch) was detected with the anti Val1744 Ab (in this and all subsequent figures). Right: Quantitation of three independent experiments. (F) HEK-293 cells, transfected as indicated (all Numb constructs were Flag-tagged and also codify for an sh-RNA sequence against endogenous Numb; EV, empty vector), were IP and IB as shown. (G) HEK-293 cells were stably transduced with Notch- NΔE (Notch-TFX; NT, not transfected) and transfected with the indicated Numb-Flag constructs (as in F). IP and IB were as shown. (H and I) MCF-7 or Cal51 cells were either transduced with Notch-NΔE (Notch-TFX; I) or not (H). Cells were treated with BIS (or mock-treated) and IP and IB as shown. In H, IP-Ctr is anti-Flag; in I, IP-Ctr is goat IgG. Data are reported ± SD (C and E) or ± SE (D). Statistical analysis was with the Student’s t test two-tailed (C and D) or with the one-sample t test (E). Source data are available for this figure: SourceData F3.

Article Snippet: Ab for immunoblot (IB) were directed against Numb (AB21, a mouse monoclonal Ab against amino acids 537–551 of hNumb [Colaluca et al., 2008]), and for the experiment in Fig. S3 B, the anti-Numb C29G11, rabbit monoclonal from Cell Signaling Technologies (Cat. 4140) was used; Vinculin (mouse monoclonal; Sigma-Aldrich, Cat. V9131); GRP94 (9G10, rat monoclonal, Cat. ADI-SPA-851; Enzo Life Sciences); Tubulin (11H10, rabbit monoclonal, Cat. 2125; Cell Signaling Technologies); p53 (1C12, mouse monoclonal, Cat. 2524; Cell Signaling Technologies, Figs. 3 E, 5, A–C, and 9 D; FL393, Santa Cruz Biotechnology, goat polyclonal, Cat. sc-6243-G, Fig. 3 F; goat polyclonal, Bio-techne, Cat. AF1355 Fig. 3 H); PKCζ (C24E6, rabbit monoclonal, Cat. 9368; Cell Signaling Technologies); p-PKCζ (H-2, mouse monoclonal, Cat. sc-271962; Santa Cruz Biotechnology); PAN-PKC (A-9, mouse monoclonal, Cat. sc-17804; Santa Cruz Biotechnology); pPAN PKC (rabbit polyclonal, Cat. 9371; Cell Signaling Technologies); Notch (5B5, rat monoclonal, Cat. 3447; Cell Signaling Technologies); Cleaved Notch1 Val1744 (D3B8, rabbit monoclonal, Cat. 4147; Cell Signaling Technologies); Flag (D6W5B, rabbit monoclonal, Cat. 2368; Cell Signaling Technologies); Par3 (Cat. 07-330; Millipore); Mdm2 (Mouse Monoclonal, Cat. Op46; Calbiochem); anti GAPDH (rabbit monoclonal, Cat. 5174; Cell Signaling Technologies); and pNumb-Ser276 (rabbit monoclonal, Cat. 4140; Cell Signaling Technologies).

Techniques: Transduction, Construct, Plasmid Preparation, Quantitation Assay, Transfection, Sequencing, Stable Transfection, Two Tailed Test

Journal: Oncotarget

Article Title: The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

doi:

Figure Lengend Snippet: Total RNA extracted from MCF7, SKBR3, MDA-MB-231 and MDA-MD-468 cell lines was subjected to qRT-PCR analysis for mRNA levels of A. Notch receptors NOTCH1, NOTCH2 B. Notch ligands JAG1, JAG2 and C. Notch Target Genes HES1 and HEY1. Data represent mean ± standard error, n = 4. D. Protein level expression of Notch1 and Jagged 1 in these cell lines was examined by Western blot analysis. β-actin was used as a loading control. E. Surface staining of Notch1 in non-permeabilized cells is shown as stacked offset histograms. Matched Isotype control from MDA-MB-468 is shown.

Article Snippet: For Western Blot we used cleaved Notch1 antibody Val1744 (Cell signaling, Danvers, MA), Jagged1 (Antibody clone H114, Santa-Cruz, CA), LC3B (Abcam). β-actin (antibody clone AC-74) was purchased from Sigma Aldrich and used as the loading control.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Staining

Journal: Oncotarget

Article Title: The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

doi:

Figure Lengend Snippet: Tissue microarray containing human breast tumors and normal breast tissues (control) were used to immunolocalize A. a2V and B. Notch1. Tumors were grouped by receptor-defined subtype. 12 sections per subtype were analyzed. Brown staining - DAB, counterstain - hematoxylin. Original magnification: 400X. Corresponding scatter dot plots show immunostaining index score ((ISIS) = Stained area score (SAS) × Immunostaining intensity score (IIS)) for C. a2V and D. Notch1. Data represent mean ± standard error, n = 12. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 between a pair of subtypes. DAB: 3,3′ Diaminobenzidine.

Article Snippet: For Western Blot we used cleaved Notch1 antibody Val1744 (Cell signaling, Danvers, MA), Jagged1 (Antibody clone H114, Santa-Cruz, CA), LC3B (Abcam). β-actin (antibody clone AC-74) was purchased from Sigma Aldrich and used as the loading control.

Techniques: Microarray, Control, Staining, Immunostaining

Journal: Oncotarget

Article Title: The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

doi:

Figure Lengend Snippet: A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after transfection. Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.

Article Snippet: For Western Blot we used cleaved Notch1 antibody Val1744 (Cell signaling, Danvers, MA), Jagged1 (Antibody clone H114, Santa-Cruz, CA), LC3B (Abcam). β-actin (antibody clone AC-74) was purchased from Sigma Aldrich and used as the loading control.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Generated, Western Blot, Gene Expression, Immunofluorescence, Construct, Luciferase, Recombinant, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: The inhibition of Notch1 signaling pathway attenuated the stemness properties of CD44v6+ LCSCs. a External dataset from starBase v3.0 project with 374 HCC samples and 50 normal samples was used to analyze the expression of Notch1. The result showed that Notch1 was higher in HCC patients’ samples than that in normal samples (1.77 fold, FDR = 0.0017). b The core components of Notch1 signaling including Notch1 receptor, cleaved Notch1 (NICD), Hey1 and Hes1 were tested in CD44v6+ LCSCs and CD44v6- HCC cells by western blot in SNU-398 cell lines. Western blot showed that CD44v6+ SNU-398 cells expressed more Notch1 signaling pathway key factors. β-actin was used as a normalized control. c Representative images of spheres and histogram analysis in indicated cells. The inhibition of Notch1 decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. d and e Transwell migration and invasion assay showed that knockdown Notch1 decreased the migration and invasion capacity of CD44v6+ cells. Scale bar, 200 μm. f Colony formation assays showed that the ability of cell proliferation and colony formation of CD44v6+ cells was inhibited when Notch1 was down regulated. g and h Efficiency of tumor formation of Notch1 shRNA cells and the corresponding controls. Right flanks of mice were injected with control CD44v6+ cells while left flanks were injected with Notch1 shRNA cells. Number of injected cells: 1 × 10 5 . n = 5. Data are expressed as mean ± SD (error bars). ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: Primary antibodies include Notch1 (CST, cat#3608), cleaved Notch1 (CST, Val1744, D3B8, cat#4147), Hes1 (CST, cat#11988), Hey1 (Abcam, ab154077), Nanog (CST, cat#4903), Sox2 (CST, cat#3579), Oct4 (CST, cat#2750), MSI2 (Abcam, ab76148), LFNG (CST, cat #66472), Numb (CST, cat #2756).

Techniques: Inhibition, Expressing, Western Blot, Control, In Vitro, Migration, Invasion Assay, Knockdown, shRNA, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 maintained the stemness properties of CD44v6+ LCSCs via activating Notch1 signaling pathway. a External dataset from starBase v3.0 project with 374 HCC samples and 50 normal samples was used to analyze the correlation of MSI2 and Notch1. The results showed that MSI2 was positively related to Notch1 in clinical-pathology ( r = 0.458, p = 8.02e-21). b Immunofluorescence images of MSI2 (red) and Notch1 (green) in HCC tissues. MSI2 was co-localized with Notch1 in HCC tissues. Scale bar, 50 μm. c Immunofluorescence images of CD44v6+ SNU-398 cells and CD44v6- cells for localization of MSI2 (red) and Notch1 (green). Histogram analysis of the relative fluorescence intensity of MSI2 and Notch1 in CD44v6+ cells and CD44v6- cells. Scale bar, 50 μm. d. Silencing MSI2 decreased the expression of Notch1 receptor and Notch1 pathway target genes in CD44v6+ SNU-398 cells. β-actin was used as a normalized control. e Western blot showed that overexpression of MSI2 increased the expression of Notch1 receptor and Notch1 pathway target genes in CD44v6- SNU-398 cells. β-actin was used as a normalized control

Article Snippet: Primary antibodies include Notch1 (CST, cat#3608), cleaved Notch1 (CST, Val1744, D3B8, cat#4147), Hes1 (CST, cat#11988), Hey1 (Abcam, ab154077), Nanog (CST, cat#4903), Sox2 (CST, cat#3579), Oct4 (CST, cat#2750), MSI2 (Abcam, ab76148), LFNG (CST, cat #66472), Numb (CST, cat #2756).

Techniques: Immunofluorescence, Fluorescence, Expressing, Control, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 activated Notch1 signaling through LFNG in CD44v6+ LCSCs. a A Notch RT 2 PCR Array was used to determine mRNA expression profiles between MSI2 shRNA and control CD44v6+ LCSCs. b Hypothesis diagram of MSI2 regulates Notch1 signaling pathway. c Relative mRNA of the most significantly regulated genes were detected by RT-PCR in MSI2 shRNA1 group and the corresponding control group. d Western blot showed that silencing MSI2 decreased the expression of LFNG in CD44v6+ cells while overexpression of MSI2 increased the expression of LFNG in CD44v6- cells. β-actin was used as a normalized control. e Efficiency of tumor formation of LFNG shRNA1 cells and the corresponding controls. Number of injected cells: 1 × 10 5 . n = 4. f Representative images of spheres and histogram analysis in indicated cells. The inhibition of LFNG decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. g The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in LFNG shRNA cells compared with corresponding controls. β-actin was used as a normalized control. The inhibition of LFNG decreased the expression of stemness-related genes in CD44v6+ LCSCs. h Silencing LFNG in CD44v6+ HCC cells decreased the expression of key components of Notch1 pathway (including Notch1, NICD, Hey1 and Hes1) but MSI2 had no significant change. β-actin was used as a normalized control. i Key components of Notch1 signaling reduction caused by MSI2 knockdown could be rescued by LFNG overexpression in CD44v6+ LCSCs. For statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: Primary antibodies include Notch1 (CST, cat#3608), cleaved Notch1 (CST, Val1744, D3B8, cat#4147), Hes1 (CST, cat#11988), Hey1 (Abcam, ab154077), Nanog (CST, cat#4903), Sox2 (CST, cat#3579), Oct4 (CST, cat#2750), MSI2 (Abcam, ab76148), LFNG (CST, cat #66472), Numb (CST, cat #2756).

Techniques: Expressing, shRNA, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Injection, Inhibition, In Vitro, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 bound to LFNG mRNA and protein directly. a Schematic representation of MSI2 molecular interaction domains for interaction with RNA and with proteins. RRM, RNA recognition motif; PPD, protein-protein binding domain. b CD44v6+ cells and CD44v6- cells were isolated from SNU-398 cells. CD44v6+ cells were transfected with MSI2 shRNA1 or corresponding control virus, CD44v6- cells were transfected with Lv MSI2 or corresponding control virus. Lysates were precipitated with anti-MSI2 antibody and then immunoblotted (WB) for LFNG. Protein expression of MSI2 and LFNG was also analyzed. c CD44v6+ cells were transfected with MSI2 shRNA1 or corresponding control virus, CD44v6- cells were transfected with Lv MSI2 or corresponding control virus. RIP assays using anti-MSI2 antibody showed that MSI2 interacted with LFNG. The results of agarose electrophoresis of the PCR products were shown. d Schematic illustration the mechanism by which MSI2 activating Notch1 signaling pathway by binding to LFNG mRNA and protein directly

Article Snippet: Primary antibodies include Notch1 (CST, cat#3608), cleaved Notch1 (CST, Val1744, D3B8, cat#4147), Hes1 (CST, cat#11988), Hey1 (Abcam, ab154077), Nanog (CST, cat#4903), Sox2 (CST, cat#3579), Oct4 (CST, cat#2750), MSI2 (Abcam, ab76148), LFNG (CST, cat #66472), Numb (CST, cat #2756).

Techniques: Protein Binding, Isolation, Transfection, Control, Virus, Expressing, Electrophoresis, Binding Assay

Journal: Cell Death & Disease

Article Title: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

doi: 10.1038/s41419-022-05178-w

Figure Lengend Snippet: A , B Primary CLL cells were cultured for 3 h with the indicated concentrations of SB216763 or DMSO as control. A Western blot analysis of NOTCH1 was performed using the anti-NOTCH1 (Val1744) and the anti-NOTCH1 (D1E11) antibodies, able to detect N1-ICD and N1-TM, respectively ( n = 8). Protein loading was assessed using an anti-GAPDH antibody. Left, the values under each blot indicate the fold change in N1-ICD and N1-TM expression in SB216763-treated cells compared with control DMSO (set to 1), normalized to GAPDH levels. GSK3β activity inhibition by SB216763 was assessed by analyzing the glycogen synthase phosphorylation at Serine 641 (pS641-GS). The values under each blot indicate the fold change in pS641-GS levels in SB216763-treated cells compared with control DMSO (set to 1), normalized to levels of total GS. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD and N1-TM, represented as fold change compared with control DMSO. * P < 0.05, ** P < 0.01 according to Wilcoxon paired test. B Flow cytometric analysis of N1-ICD performed using the mouse anti-NOTCH1 (mN1A)-PE antibody ( n = 6). Left, results are represented as the percentage of N1-ICD positive cells. N1-ICD positive gate was set based on staining with PE-mouse IgG isotype control. One CLL sample is shown. Right, box and whisker plots with data points of the percentage of N1-ICD positive cells, represented as fold change compared with control DMSO set to 1. * P < 0.05 according to Wilcoxon paired test. C CLL cells were transfected with control siRNA (siCtrl) or GSK3β siRNA (siGSK3β) ( n = 8). Left, N1-ICD expression was analyzed as in panel A. Protein loading was assessed using an anti-GAPDH antibody. Silencing efficiency was assessed by Western blot analysis of GSK3β. The values under each blot indicate the fold change in N1-ICD and GSK3β levels in siGSK3β cells compared with siCtrl cells (set to 1), normalized to GAPDH levels. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD, represented as fold change compared with siCtrl cells. ** P < 0.01 according to Wilcoxon paired test. D CLL cells were transiently transfected with the pcDNA3.1 empty vector as control or the pcDNA3 plasmid containing the constitutively active GSK3β (GSK3β S9A) ( n = 6). Left, N1-ICD expression was analyzed as in panel A. Protein loading was assessed using an anti - GAPDH antibody. Transfection efficiency was assessed by Western blot analysis of GSK3β. The values under each blot indicate the fold change in N1-ICD and GSK3β expression in S9A-transfected cells compared with empty vector-transfected cells (set to 1), normalized to GAPDH levels. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD, represented as fold change compared with empty vector-transfected cells. * P < 0.05 according to Wilcoxon paired test.

Article Snippet: The beads were pelleted, and the lysates were recovered and incubated at 4 °C overnight with 6 μg of the rabbit monoclonal anti-NOTCH1 antibody (Val1744; clone D3B8, Cell Signaling Technology) or normal rabbit IgG antibody as negative control (Cell Signaling Technology).

Techniques: Cell Culture, Control, Western Blot, Expressing, Activity Assay, Inhibition, Phospho-proteomics, Whisker Assay, Staining, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

doi: 10.1038/s41419-022-05178-w

Figure Lengend Snippet: A Box and whisker plots with data points of real-time PCR analysis of NOTCH1, HES1 and DELTEX (DTX) mRNA in CLL cells cultured for 3 h with 5 μM SB216763 or DMSO as control ( n = 12). mRNA levels were normalized to GAPDH and represented as fold change using control cells as a reference. * P < 0.05; ns, not significant, according to Wilcoxon paired test. B , C After pretreatment with 5 μM SB216763 or DMSO for 1.5 h, CLL cells were treated ( T = 0) with 50 μg/ml CHX and harvested at the indicated times for Western blot analysis of N1-ICD and GAPDH, as a loading control ( n = 6). B The values under the blots relative to each treatment indicate the fold change in N1-ICD expression at the different time points compared with the respective T = 0 (set to 1), normalized to GAPDH levels. Three CLL samples are shown. C N1-ICD bands were quantified by densitometry analysis, normalized to GAPDH and represented as percentage of T = 0 value set to 100%. Data are presented as the mean ± SD of 6 CLL samples. * P < 0.05 according to Wilcoxon paired test. D CLL cells were cultured for 18 h with 5 μM SB216763 or DMSO as control ( n = 8). Cell viability was measured by MTS assay. Box and whisker plots with data points, expressed as optical density (OD) values, are shown. ** P < 0.01 according to Wilcoxon paired test.

Article Snippet: The beads were pelleted, and the lysates were recovered and incubated at 4 °C overnight with 6 μg of the rabbit monoclonal anti-NOTCH1 antibody (Val1744; clone D3B8, Cell Signaling Technology) or normal rabbit IgG antibody as negative control (Cell Signaling Technology).

Techniques: Whisker Assay, Real-time Polymerase Chain Reaction, Cell Culture, Control, Western Blot, Expressing, MTS Assay

Journal: Cell Death & Disease

Article Title: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

doi: 10.1038/s41419-022-05178-w

Figure Lengend Snippet: A N1-ICD was immunoprecipitated (IP) from whole-cell extracts of CLL cells, and the IP lysates were analyzed by Western blot with the anti-NOTCH1 (Val1744) to confirm IP of N1-ICD, and with the anti-GSK3β antibody to detect GSK3β/N1-ICD interaction ( n = 3). One representative CLL is shown. B Confocal microscopy images of subcellular localization of GSK3β in a representative CLL sample. CLL cells ( n = 3) were stained with the anti-GSK3β antibody (red) and with DAPI for nuclei (blue) and then analyzed by confocal microscopy, with a 63x oil immersion and 1.4 NA objective; scale bar, 10 μm. C , D PLA was performed by using rabbit anti-NOTCH1 (Val1744) and mouse anti-GSK3β antibodies to detect GSK3β/N1-ICD interactions in CLL cells cultured for 1.5 h with 5 µM SB216763 or DMSO ( C ; n = 3), and by using rabbit anti-NOTCH1 (Val1744) and mouse anti-ubiquitin antibodies to detect N1-ICD/Ubiquitin interactions in CLL cells cultured with 5 µM SB216763 or DMSO for 1.5 h, and with 10 µM MG132 for additional 4 h ( D ; n = 3). Nuclei were stained with DAPI. In the confocal microscopy images, red spots indicate GSK3β/N1-ICD ( C ) and N1-ICD/Ubiquitin ( D ) interactions. Images were acquired by using confocal microscopy with a 63x oil immersion and 1.4 NA objective; scale bar, 10 μm. One representative CLL is shown. In the bottom panel ( C ) and in the right panel ( D ), bar graphs ± SEM show quantitative analysis of the PLA signals of three samples. *** *P < 0.0001; * *P < 0.01; *P < 0.05 according to unpaired Student’s t- test.

Article Snippet: The beads were pelleted, and the lysates were recovered and incubated at 4 °C overnight with 6 μg of the rabbit monoclonal anti-NOTCH1 antibody (Val1744; clone D3B8, Cell Signaling Technology) or normal rabbit IgG antibody as negative control (Cell Signaling Technology).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Cell Culture, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

doi: 10.1038/s41419-022-05178-w

Figure Lengend Snippet: A CLL cells were cultured for 6 h with 5 μM AKTiX (AiX) or complete medium as control ( n = 10). Western blot analysis of NOTCH1 was performed using the anti-NOTCH1 (Val1744) and the anti-NOTCH1 (D1E11) antibodies, able to recognize N1-ICD and N1-TM, respectively. Protein loading was assessed using an anti-GAPDH antibody. Left, the values under each blot indicate the fold change in N1-ICD and N1-TM levels in AiX-treated cells compared with control cells (set to 1), normalized to GAPDH levels. AKT Inhibition by AiX was verified by analyzing AKT phosphorylation at Serine 473 (pS473-AKT). The effect of AiX on GSK3β inactivation was assessed by analyzing pS9-GSK3β levels. The values under each blot indicate the fold change in pS473-AKT and pS9-GSK3β levels in AiX-treated cells compared with control cells (set to 1), normalized to levels of total AKT and total GSK3β, respectively. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD and N1-TM, represented as fold change compared with controls. ** P < 0.01; ns, not significant according to Wilcoxon paired test. B CLL cells were cultured for 1.5 h with 5 µM SB216763 or DMSO and for further 6 h with 5 µM AiX ( n = 6). Western blot analysis of N1-ICD, pS9-GSK3β, total GSK3β and GAPDH was performed as in panel A. Left, the values under the blots indicate the fold change in N1-ICD and pS9-GSK3β levels in cells treated with AiX alone or AiX plus SB216763, compared with control cells (set to 1), normalized to levels of GAPDH and total GSK3β, respectively. Three CLL samples are shown. Right, box and whisker plots with data points of densitometry analysis of N1-ICD, represented as fold change compared with control. * P < 0.05 according to Wilcoxon paired test. C CLL cells were pretreated for 2 h with 10 μM MG132 or DMSO, and then cultured for further 6 h with or without 5 μM AiX ( n = 8). Western blot analysis of N1-ICD, pS9-GSK3β, total GSK3β and GAPDH was performed as in panel A. Top, the values under the blots indicate the fold change in N1-ICD and pS9-GSK3β levels in cells treated with AiX, MG132, or AiX plus MG132, compared with control cells (set to 1), normalized to levels of GAPDH and total GSK3β, respectively. Three CLL samples are shown. Vertical lines inserted in CLL1 and CLL30 blots indicate repositioned gel lanes. Bottom, box and whisker plots with data points of densitometry analysis of N1-ICD, represented as fold change compared with control. ** P < 0.01; ns, not significant according to Wilcoxon paired test. D , E CLL cells were cultured with or without different concentrations of AiX (2.5, 5 and 10 µM) or SB216763 (2.5, 5 and 10 µM) alone or in combinations ( n = 6). After 18 h, cell viability was measured by MTS assay. D Bar graphs with data points of cell viability (mean ± SD) in treated cells compared with untreated controls, set to 100%. * P < 0.05 according to Wilcoxon paired test. E The antagonism between SB216763 and AiX was calculated by using the SynergyFinder web application and the results were produced with ZIP Synergy model (green indicates an antagonistic effect, white an additive effect, and red a synergistic effect).

Article Snippet: The beads were pelleted, and the lysates were recovered and incubated at 4 °C overnight with 6 μg of the rabbit monoclonal anti-NOTCH1 antibody (Val1744; clone D3B8, Cell Signaling Technology) or normal rabbit IgG antibody as negative control (Cell Signaling Technology).

Techniques: Cell Culture, Control, Western Blot, Inhibition, Phospho-proteomics, Whisker Assay, MTS Assay, Produced

Journal: Cell Death & Disease

Article Title: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

doi: 10.1038/s41419-022-05178-w

Figure Lengend Snippet: A A schematic outline of the treatment schedule is shown. E μ -TCL1 cells were transplanted into C57BL/6 mice by intravenous (i.v.) injection. Twenty-eight days after transplantation (day 0), treatment started with DT-061 (5 mg/kg once daily for 28 days via oral gavage; n = 3) or vehicle ( n = 3). Peripheral blood (PB) was harvested at the start of treatment (day 0), and at day +14 and day +28 from the start of the treatment. At day +28, mice were sacrificed, and spleen and bone marrow were collected. B The bar graphs with data points indicate the percentage of CD19 + /CD5 + cells in PB from DT-061- and vehicle-treated mice, determined by flow cytometry. Data are presented as the mean ± SD of 3 mice per group. * P < 0.05 according to unpaired Student’s t -test. C , D The bar graphs with data points indicate the number (left) and the percentage (middle) of CD19 + /CD5 + cells in the spleen ( C ) and bone marrow ( D ) from DT-061- and vehicle-treated mice, determined by flow cytometry. Data are presented as the mean ± SD of 3 mice per group. * P < 0.05; *** P < 0.001 according to unpaired Student’s t -test. One representative dot plot of CD19/CD5 staining relative to each treatment is shown (right). E , F Left, bar graphs with data points indicate the percentage of viable Annexin V − (An V − ) cells in CD19 + /CD5 + sorted from the spleen ( E ) and bone marrow ( F ) of DT-061- and vehicle-treated mice. Data are presented as the mean ± SD of 3 mice per group. * P < 0.05 according to unpaired Student’s t- test. Middle, Western blot analysis of N1-ICD in CD19 + /CD5 + cells sorted from the spleen ( E ) and bone marrow ( F ) of DT-061- and vehicle-treated mice performed using the anti-NOTCH1 Val1744 antibody. Right, bar graphs with data points of densitometric analysis of N1-ICD are shown. * P < 0.05; ** P < 0.01 according to unpaired Student’s t -test.

Article Snippet: The beads were pelleted, and the lysates were recovered and incubated at 4 °C overnight with 6 μg of the rabbit monoclonal anti-NOTCH1 antibody (Val1744; clone D3B8, Cell Signaling Technology) or normal rabbit IgG antibody as negative control (Cell Signaling Technology).

Techniques: Injection, Transplantation Assay, Flow Cytometry, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

doi: 10.1038/s41419-022-05178-w

Figure Lengend Snippet: Constitutive NOTCH1-ICD levels and CLL cell survival are sustained by GSK3β inactivation, due to an impaired PP2A activity and a high AKT activation, which both induce S9-GSK3β phosphorylation (pS9-GSK3β). The increase in PP2A activity induced by the highly specific activator DT-061 or the inhibition of AKT with the AKTiX inhibitor enhance GSK3β activity, leading to a decrease in NOTCH1-ICD levels with a concomitant reduction in CLL cell survival.

Article Snippet: The beads were pelleted, and the lysates were recovered and incubated at 4 °C overnight with 6 μg of the rabbit monoclonal anti-NOTCH1 antibody (Val1744; clone D3B8, Cell Signaling Technology) or normal rabbit IgG antibody as negative control (Cell Signaling Technology).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Inhibition