Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: BMAL1-deletion upregulates ceramide level in AML cells (A) qPCR validation of BMAL1 expression in peripheral blood mononuclear cells from healthy donors (HD [ n = 6])and bone marrow cells from newly diagnosed ( n = 9), remission ( n = 9), and relapse ( n = 9) stage AML patients. (B) Kaplan-Meier curves of control, BMAL1-OE or BMAL1 shRNA HL60 AML tumor-bearing mice ( n = 6 mice/group). (C) Kaplan-Meier curve indicating overall survival, event free survival and post progression survival of patients based on AML BMAL1 expression from TCGA database. AML patients were categorized into high-BMAL1 and low-BMAL1 mRNA expression groups utilizing the minimum p value approach. (D) Lipid-lipid interaction networks. Lipid molecule pairs with a correlation coefficient of |r|>0.8 and p < 0.05. Chord diagram (left), the points of the inner circle links represent significantly different lipid molecules, while the outer circle arcs represent lipid subclasses. Colored lines depict the correlation among lipid molecules within the subclasses. Dark gray lines represent the correlation between different subclasses. Network diagram (right), the nodes represent significantly different lipid molecules, with larger nodes indicate higher degrees. The color of the lines represents the correlation, where purple indicates positive correlation and green indicates negative correlation. Thicker lines represent stronger correlations. (E and F) The level of ceramide and viability analysis of HL60 and MOLM13 cells with control or BMAL1 shRNA (versus blank control group). Cells were treated with or without RSL3 (0.5 μM) for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (A), Mann-Whitney U test. (B and C), log rank test p values are shown. (E and F), two-way ANOVA using Dunnett multiple comparisons test with adjusted p values are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: Biomarker Discovery, Expressing, Control, shRNA, MANN-WHITNEY

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: Ceramide upregulation sensitizes AML cells to lipid peroxidation and ferroptosis (A–C) Viability analysis, the level of MDA and the lipid ROS of HL60 and MOLM13 cells with control or BMAL1-OE (versus blank control group). Scale bar: 100 μm. Cells were treated with RSL3 (0.5 μM) in the presence or absence of C2-ceramide (C2-Cer, 5.0 μM) or ferrostain-1 (Fer-1, 0.5 μM) for 24 h. (D–F) Viability analysis, the level of MDA and the lipid ROS of HL60 and MOLM13 cells with control or BMAL1 shRNA (versus blank control group). Cells were treated with RSL3 (0.5 μM) in the presence or absence of myriocin (ISP-1, 1.0 μM) or ferrostain-1(Fer-1, 0.5 μM) for 24 h, Scale bar: 100 μm. Before treating with RSL3, the cells were pre-treated with ISP-1 for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (A, B, D, and E), two-way ANOVA using Šídák multiple comparisons test with adjusted p values are shown. (C and F), one-way ANOVA using Tukey multiple comparisons test with adjusted p values are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: Control, shRNA

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: ASAH2 is the key mediator in the elevation of ceramide levels induced by BMAL1 knockdown (A) The major metabolites and the key sphingolipid metabolic genes associated with ceramide metabolism in KEGG-based sphingolipid metabolism network. 3-KS, 3-ketosphinganine; Sph, sphinganine; DhCer, dihydroceramide; Cer, ceramide; SM, sphingomyelin; GalCer, galactosylceramide; Psy, psychosine; Spho, sphingosine; C1P, ceramide-1-phosphate; S1P, sphingosine-1-phosphate. (B) Table shows the average fold change and statistical analysis of 19 sphingolipid metabolic gene expression between BMAL1 shRNA/BMAL1-OE and their parental WT cell lines of HL60 and MOLM13 cells. Cells were treated with RSL3 (0.5 μM) for 24 h. (C) Western blot analysis showing indicated protein expression in HL60 and MOLM13 cells with control, BMAL1-OE, or BMAL1 shRNA. Cells were treated with RSL3 (0.5 μM) for 24 h. (D) The lipid ROS were assayed of HL60 and MOLM13 cells with control, BMAL1-OE or BMAL1-OE+ASAH2 shRNA. Cells were treated with RSL3 (0.5 μM) for 24 h, Scale bar: 100 μm. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (B), two-way ANOVA using Šídák multiple comparisons test with adjusted p values is shown. (C), two-way ANOVA using Dunnett multiple comparisons test with adjusted p values is shown. (D), one-way ANOVA using Tukey multiple comparisons test with adjusted p values is shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: Knockdown, Gene Expression, shRNA, Western Blot, Expressing, Control

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: IKZF2 mediates BMAL1's regulation of ASAH2 (A) ChIP-qPCR assays were performed using anti-BMAL1, FLAG, or IgG, with primer pairs targeting the ASAH2 promoter in 293T cells. (B) The overlap gene image of BMAL1-regulated genes and potential transcription factors of ASAH2 . (C) Tables show the average fold change and statistical analysis of 24 gene expression between BMAL1 shRNA/BMAL1-OE and their parental WT cell lines of HL60 and MOLM13 cells. Cells were treated with RSL3 (0.5 μM) for 24 h. (D–F) qPCR, western blot, and the level of ceramide analysis in HL60 and MOLM13 cells with control, IKZF2-OE, or IKZF2 shRNA. Cells were treated with or without RSL3 (0.5 μM) for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (A and C), two-way ANOVA using Šídák multiple comparisons test with adjusted p values are shown. (D–F), two-way ANOVA using Dunnett multiple comparisons test with adjusted p values are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: ChIP-qPCR, Gene Expression, shRNA, Western Blot, Control

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: IKZF2 mediates BMAL1's regulation of ASAH2 (A) ChIP-qPCR assays were performed using anti-BMAL1, FLAG, or IgG, with primer pairs targeting the IKZF2 promoter in 293T cells. (B) ChIP-qPCR assays were performed using anti-IKZF2, FLAG, or IgG, with primer pairs targeting the ASAH2 promoter in 293T cells. (C) Luciferase assays of 293T cells co-transfected with a luciferase reporter containing a full-length human IKZF2 (left) or ASAH2 (right) promoter sequence and an escalating amount of BMAL1 (left) or IKZF2 (right) overexpression plasmid. (D) Luciferase assays were performed on 293T cells co-transfected with a luciferase vector carrying either a full-length WT IKZF2 (left) or ASAH2 (right) promoter sequence, or a promoter sequence containing a mutated proximal or distal BMAL1 (left) or IKZF2 (right) binding site, together with 160 ng of BMAL1 (left) or 200 ng of IKZF2 (right) overexpression plasmid. (E–G) qPCR, western blot, and the levels of ceramide analysis in HL60 and MOLM13 cells with control, BMAL1-OE or BMAL1-OE +IKZF2-OE. Cells were treated with or without RSL3 (0.5 μM) for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (A and B), two-way ANOVA using Šídák multiple comparisons test with adjusted p values are shown. (C and D), one-way ANOVA using Dunnett multiple comparisons test with adjusted p values are shown. (E–G), two-way ANOVA using Tukey multiple comparisons test with adjusted p values are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: ChIP-qPCR, Luciferase, Transfection, Sequencing, Over Expression, Plasmid Preparation, Binding Assay, Western Blot, Control

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: GPX4 degradation promotes ceramide upregulation-mediated ferroptosis (A and B) The lipid ROS were assayed of HL60 and MOLM13 cells with vector, BMAL1-OE, BMAL1 shRNA, BMAL1-OE+IKZF2-OE, or BMAL1 +IKZF2 shRNA. Cells were treated with or without RSL3 (0.5 μM) for 24 h, Scale bar: 100 μm. (C) Western blot analysis showing indicated protein expression in HL60 and MOLM13 cells with control, BMAL1-OE, or BMAL1-OE + ASAH2 shRNA. Cells were treated with or without RSL3 (0.5 μM) for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (A and B), one-way ANOVA using Tukey multiple comparisons test with adjusted p values are shown. (C), two-way ANOVA using Tukey multiple comparisons test with adjusted p values is shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: Plasmid Preparation, shRNA, Western Blot, Expressing, Control

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: BMAL1 depletion boosts AML cell sensitivity to sorafenib (A) Viability analysis of HL60 and MOLM13 cells with control or BMAL1 shRNA (versus blank control group). Cells were treated with sorafenib (1.25, 0.4 μM) in the presence or absence of myriocin (ISP-1, 1.0 μM) or ferrostain-1 (Fer-1, 0.5 μM) for 24 h. Before treating with sorafenib, the cells were pre-treated with ISP-1 for 24 h. (B and C) The level of ceramide and viability analysis of HL60 and MOLM13 cells with control, BMAL1 shRNA or BMAL1+ IKZF2 shRNA (versus blank control group). Cells were treated with or without sorafenib (1.25, 0.4 μM) for 24 h. (D and E) The level of ceramide and viability analysis of HL60 and MOLM13 cells with control, BMAL1-OE or BMAL1-OE+ASAH2 shRNA (versus blank control group). Cells were treated with or without sorafenib (1.25, 0.4 μM) for 24 h. Data are presented as mean ± SD, n = 3 biologically independent samples. Statistical tests used: (A), two-way ANOVA using Šídák multiple comparisons test with adjusted p values is shown. (B–E), two-way ANOVA using Tukey multiple comparisons test with adjusted p values are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: Control, shRNA

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet: The BMAL1-IKZF2-ASAH2 pathway enhancing ferroptosis-mediated AML suppression in vivo (A) The schematic diagram of the complete mouse experiment procedure. (B–F) The level of 4-HNE (B), MDA (C), PTGS2 mRNA (D), and ceramide (F) in the hepatic tumors tissue, and the level of serum HMGB1 (E) were assayed at the endpoint time after treatment ( n = 4 mice/group). (G) The weight of mice at the endpoint time after treatment ( n = 4 mice/group). (H) The GFP+ leukemia cells in peripheral blood of HL60 leukemia recipient mice at indicated days in treatment groups ( n = 4 mice/group). (I) The weight of liver and spleen in mice at the endpoint time after treatment ( n = 4 mice/group). (J) HE staining of liver of mice at the endpoint time after treatment. Scale bare: 100 μm. Data are presented as mean ± SD. Statistical tests used: (B–G and I), one-way ANOVA using Tukey multiple comparisons test with adjusted p values are shown. (H), two-way ANOVA using Tukey multiple comparisons test with adjusted p values is shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: In Vivo, Staining

Journal: iScience

Article Title: BMAL1-depletion remodels ceramide metabolism to regulate ferroptosis and sorafenib chemosensitivity in acute myeloid leukemia

doi: 10.1016/j.isci.2025.112054

Figure Lengend Snippet:

Article Snippet: C2 Ceramide , MCE , Cat#HY-101180.

Techniques: Bicinchoninic Acid Protein Assay, Lysis, Transfection, Staining, SYBR Green Assay, CCK-8 Assay, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay, Software, Flow Cytometry

Journal: FEBS letters

Article Title: Sphingosine-1-phosphate stimulates cortisol secretion.

doi: 10.1016/s0014-5793(02)03882-6

Figure Lengend Snippet: Fig. 1. Stimulation of cortisol secretion by di¡erent lipid agonists. Cells were stimulated for 2 h with increasing concentrations of Sph- 1-P (solid circles), sphingosine (empty circles), N-octanoyl-Sph-1-P (squares), or LPA (triangles). For experimental details see Section 2. Results are expressed as fold stimulation relative to incubations with vehicle, and show the mean T S.E.M. of four independent ex- periments, except for the value at 5 WM Sph-1-P, which is the mean T S.E.M. of 36 experiments. Basal value of cortisol secretion was 7.6 T 1.0 ng/mg protein (mean T S.E.M., n = 48).

Article Snippet: Sphingosine, Sph-1-P, N-acetylsphingosine (C2-ceramide), dihydro-C2-ceramide, N-octanoyl-Sph-1P, and phosphatidylethanol standard were from Avanti Polar Lipids (Alabaster, AL, USA).

Techniques:

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Schematic representation of the sequence organization of OsCERK . The sizes of the 5′-UTR (white), exons (black) and 3′-UTR (grey) are indicated by numbers below the boxes. The sizes of introns are shown above. (B) Phylogenetic analysis of CERKs and the related enzymes sphingosine kinase (SPK)s, and diacylglycerol kinase (DAGK)s. The architecture of each gene was obtained from SMART using the architecture analysis function, and conserved domains (letters a–d) are indicated as follows: a, Pleckstrin homology domain (PH domain; accession number SM00233); b, Diacylglycerol kinase catalytic domain (DAGKc; SMART accession number SM00046); c, segments of low compositional complexity determined by the program; d, diacylgycerol kinase catalytic domain (DAGK_cat; Pfam accession number PF00781). Detailed sequences information is described in . AtCERK is ACD5. (C) CLUSTAL W alignment of CERK from various organisms showing the highly conserved CXXXCXXC motif. (D) RT-PCR expression analysis of OsCERK in leaves, stem and roots of 2-week-old seedlings. β-actin was used as a control. (E) CERK activity of rice extracts. Crude plant extracts made from leaves and roots of rice, respectively, were incubated at 40°C for 30 min as described in . The reaction mixture (1 µl) was resolved on a TLC plate. Purified OsCERK recombinant protein was used as the positive control (+CK). For the negative control (−CK) no extract was added. (F) Comparison of recombinant OsCERK and rACD5. The recombinant protein (1.0 µg) was added in 100 µl reaction system, and incubated at 40°C for 30 min. –CK, no recombinant protein was added. Arrows in (E and F) indicated the formed product C6-NBD-ceramide-1-phosphate (C6-NBD-Cer-1P).

Article Snippet: C2 ceramide-1-phosphate (C2-1-P) and N, N-dimethylsphingosine (DMS) were from Avanti Polar Lipids (Alabaster, AL, USA), and dissolved in ethanol.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Incubation, Purification, Recombinant, Positive Control, Negative Control

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Effects of cycloheximide (CHX) on C6 ceramide-induced cell death. (B) Effects of K252a on C6 ceramide-induced cell death. (C) Effects of C2 ceramide-1-phosphate on C2 ceramide induced cell death. Protoplasts were treated with less than 0.15% solvent or the indicated reagent. Percentage of surviving cells was estimated by FDA staining. These assays were repeated three times with similar results. Letters indicate that values are different using Fisher's PLSD test ( P <0.05). Bars show standard deviations.

Article Snippet: C2 ceramide-1-phosphate (C2-1-P) and N, N-dimethylsphingosine (DMS) were from Avanti Polar Lipids (Alabaster, AL, USA), and dissolved in ethanol.

Techniques: Staining

Journal: Clinical Science (London, England : 1979)

Article Title: Acid sphingomyelinase promotes SGK1-dependent vascular calcification

doi: 10.1042/CS20201122

Figure Lengend Snippet: ( A ) Representative confocal microscopy images showing ceramide levels in HAoSMCs following treatment for 5 min with control (CTR), bacterial sphingomyelinase (SMase) or β-glycerophosphate (Pi) alone and together with bacterial sphingomyelinase (SMase). Ceramide: green labeling, nuclei: magenta labeling. Scale bar: 50 μm. ( B ) Representative histograms of flow cytometry analysis and scatter dot plots and arithmetic means ± SEM ( n =6; arbitrary units, a.u.) of normalized ceramide-dependent fluorescence intensity in HAoSMCs following treatment for 5 min with control (CTR), bacterial sphingomyelinase (SMase) or β-glycerophosphate (Pi) alone and together with bacterial sphingomyelinase (SMase) or amitriptyline (Ami). **( P <0.01), ***( P <0.001) significant compared with control HAoSMCs; †††( P <0.001) significant compared with Pi-treated HAoSMCs.

Article Snippet: HAoSMCs were treated with 0.01 U/ml sphingomyelinase ( Staphylococcus aureus ) (Enzo Life Sciences) [ ], 10 µM C2-ceramide (stock in DMSO, Enzo Life Sciences) [ ], 2 mM β-glycerophosphate (Sigma–Aldrich) [ ], 5 µM amitriptyline (Sigma–Aldrich) [ ], 5 µM fendiline (stock in DMSO, Santa Cruz Biotechnology), 1 µM LY294002 (stock in DMSO, Enzo Life Sciences) and/or 100 nM wortmannin (stock in DMSO, Enzo Life Sciences) [ ].

Techniques: Confocal Microscopy, Labeling, Flow Cytometry, Fluorescence

Journal: Clinical Science (London, England : 1979)

Article Title: Acid sphingomyelinase promotes SGK1-dependent vascular calcification

doi: 10.1042/CS20201122

Figure Lengend Snippet: ( A–C ) Scatter dot plots and arithmetic means ± SEM ( n =6; arbitrary units, a.u.) of MSX2 (A), CBFA1 (B) and ALPL (C) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR), C2-ceramide (C2-cer) or β-glycerophosphate (Pi) alone and together with C2-ceramide (C2-cer). ( D ) Scatter dot plots and arithmetic means ± SEM ( n =10, a.u.) of normalized ALP activity in HAoSMCs following treatment for 7 days with control (CTR), C2-ceramide (C2-cer) or β-glycerophosphate (Pi) alone and together with C2-ceramide (C2-cer). ( E ) Representative images showing Alizarin Red staining in HAoSMCs following treatment for 11 days with control (CTR), C2-ceramide (C2-cer) or calcification medium (Calc.) alone and together with C2-ceramide (C2-cer). The calcified areas are shown as red. ( F ) Scatter dot plots and arithmetic means ± SEM ( n =4; µg/mg protein) of calcium content in HAoSMCs following treatment for 11 days with control (CTR), C2-ceramide (C2-cer) or calcification medium (Calc.) alone and together with C2-ceramide (C2-cer). *( P <0.05), **( P <0.01), ***( P <0.001) significant compared with control HAoSMCs; †( P <0.05), ††( P <0.01), †††( P <0.001) significant compared with Pi/Calc.-treated HAoSMCs.

Article Snippet: HAoSMCs were treated with 0.01 U/ml sphingomyelinase ( Staphylococcus aureus ) (Enzo Life Sciences) [ ], 10 µM C2-ceramide (stock in DMSO, Enzo Life Sciences) [ ], 2 mM β-glycerophosphate (Sigma–Aldrich) [ ], 5 µM amitriptyline (Sigma–Aldrich) [ ], 5 µM fendiline (stock in DMSO, Santa Cruz Biotechnology), 1 µM LY294002 (stock in DMSO, Enzo Life Sciences) and/or 100 nM wortmannin (stock in DMSO, Enzo Life Sciences) [ ].

Techniques: Expressing, Activity Assay, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: Acid sphingomyelinase promotes SGK1-dependent vascular calcification

doi: 10.1042/CS20201122

Figure Lengend Snippet: ( A ) Representative Western blots and scatter dot plots and arithmetic means ± SEM ( n =5; arbitrary units, a.u.) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment for the indicated times with bacterial sphingomyelinase (SMase). ( B ) Representative Western blots and scatter dot plots and arithmetic means ± SEM ( n =5; a.u.) of normalized SGK1/GAPDH protein ratio in HAoSMCs following treatment for the indicated times with C2-ceramide (C2-cer). *( P <0.05) significant compared with control HAoSMCs.

Article Snippet: HAoSMCs were treated with 0.01 U/ml sphingomyelinase ( Staphylococcus aureus ) (Enzo Life Sciences) [ ], 10 µM C2-ceramide (stock in DMSO, Enzo Life Sciences) [ ], 2 mM β-glycerophosphate (Sigma–Aldrich) [ ], 5 µM amitriptyline (Sigma–Aldrich) [ ], 5 µM fendiline (stock in DMSO, Santa Cruz Biotechnology), 1 µM LY294002 (stock in DMSO, Enzo Life Sciences) and/or 100 nM wortmannin (stock in DMSO, Enzo Life Sciences) [ ].

Techniques: Western Blot

Journal: Clinical Science (London, England : 1979)

Article Title: Acid sphingomyelinase promotes SGK1-dependent vascular calcification

doi: 10.1042/CS20201122

Figure Lengend Snippet: ( A–C ) Scatter dot plots and arithmetic means ± SEM ( n =6; arbitrary units, a.u.) of MSX2 (A), CBFA1 (B) and ALPL (C) relative mRNA expression in HAoSMCs following transfection with negative control siRNA (Neg.si) or SGK1 siRNA (SGK1si) and treatment for 24 h with control or bacterial sphingomyelinase (SMase). ( D–F ) Scatter dot plots and arithmetic means ± SEM ( n =6; a.u.) of MSX2 (D), CBFA1 (E) and ALPL (F) relative mRNA expression in HAoSMCs following transfection with negative control siRNA (Neg.si) or SGK1 siRNA (SGK1si) and treatment for 24 h with control or C2-ceramide (C2-cer). *( P <0.05), **( P <0.01), ***( P <0.001) significant compared with Neg.si-transfected HAoSMCs; †( P <0.05), ††( P <0.01) significant compared with Negsi-transfected and SMase/C2-cer-treated HAoSMCs.

Article Snippet: HAoSMCs were treated with 0.01 U/ml sphingomyelinase ( Staphylococcus aureus ) (Enzo Life Sciences) [ ], 10 µM C2-ceramide (stock in DMSO, Enzo Life Sciences) [ ], 2 mM β-glycerophosphate (Sigma–Aldrich) [ ], 5 µM amitriptyline (Sigma–Aldrich) [ ], 5 µM fendiline (stock in DMSO, Santa Cruz Biotechnology), 1 µM LY294002 (stock in DMSO, Enzo Life Sciences) and/or 100 nM wortmannin (stock in DMSO, Enzo Life Sciences) [ ].

Techniques: Expressing, Transfection, Negative Control

Journal:

Article Title: ?4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium

doi:

Figure Lengend Snippet: A: Confocal microscopy of cytokeratin 18 and NFκB p65. Control S−1 acini in rBM treated for 1 hr with TNF-α (100 nM), Trail peptide (1 µg/ml), or etoposide (50 µM) show cytoplasmic (cytokeratin 18; FITC) to nuclear translocation of NFκB p65 (Texas red), whereas acini treated with C2-ceramide (5 µM) do not. B: Cell viability calculated for control S−1 cells or S−1 cells expressing a proteolytically resistant mutant IκBα grown in rBM to form acini and then treated as described in A in the presence or absence of the multicatalytic proteosome inhibitor MG 132 (5 µM). Results are mean ± SEM of 3–4 separate experiments with 200–600 cells scored in each.

Article Snippet: Apoptosis was initiated by direct receptor trimerization using the FAS receptor antibody IgM Ch-11 (1–2 µg/ml); by receptor ligation with CD95 (1–2 µg/ml) followed by secondary mAb-induced clustering; and by treatment with recombinant, purified Trail (Apo2L, 1–4 µg/ml; BioMol Research Laboratories Inc.), recombinant TNF-α (10–100 nM; R&D Systems Inc.), the topoisomerase II inhibitor, etoposide (10–100 µM in DMSO; TopoGen Inc.), the microtubule agent paclitaxol (20–120 nM; Sigma), the actin microfilament drug cytochalasin B (1–2 µg/ml; Sigma), the membrane permeable N-acetylsphinogine analog C2-ceramide (0.5–5 µM; BioMol), or the membrane-permeable nonactive N-acetylsphinogine analog C2-dihydroxceramide (0.5–5 µM; BioMol).

Techniques: Confocal Microscopy, Translocation Assay, Expressing, Mutagenesis