Journal: JCI Insight

Article Title: Upregulation of acid ceramidase contributes to tumor progression in tuberous sclerosis complex

doi: 10.1172/jci.insight.166850

Figure Lengend Snippet: 621-101 (TSC2-) and 621-103 (TSC2+) cells were treated with an ASAH1 inhibitor, 17a ( A ) or carmofur ( B ), with indicated concentrations for 72 hours. Cell viability was measured using MTT assay ( n = 6/treatment group). Error bars show SEM. ( C ) TSC2-null 621-101 cells were transfected with 3 independent ASAH1 siRNAs or control siRNA for 48 hours. siRNA knockdown efficiency was determined by qRT-PCR ( n = 3/group) and ( D ) immunoblotting analysis. Fold-change of ASAH1/β-actin was determined using densitometry analysis. β-Actin was used as a loading control. ( E ) Cell viability was assessed 48 hours after ASAH1 siRNA transfection in 621-101 cells using MTT assay ( n = 12/treatment group). ( F ) Cells were treated with ceramide, then stained with Annexin V: FITC Apoptosis Detection Kit (BD). Cell death was analyzed by flow cytometry ( n = 3). ( G ) The percentage of apoptotic (annexin V + ) cells was determined (percentage of Annexin V: FITC-positive cells in total cell number). ( H ) 621-101 cells were infected with lentiviruses containing shRNA for vector pLKO.1, or ASAH1 , then selected with puromycin for 2 weeks. shRNA knockdown efficiency was determined by immunoblotting. β-Actin was used as a loading control. ( I ) Viability of 621-101 cells transduced with pLKO.1 or ASAH1 shRNA was measured by MTT assay ( n = 8–16/treatment group). ( J ) Stable cells were harvested, then stained with Annexin V: FITC Apoptosis Detection Kit. Cell death was analyzed by flow cytometry ( n = 3). ( K ) The percentage of apoptotic (annexin V + ) cells was determined (percentage of Annexin V: FITC-positive cells in total cell number). ( L ) Cell death was measured using PI exclusion assay. Relative cell death was compared between pLOK.1 and shRNA- ASAH1 cells. ( A – C , E , G , I , K , and L ) ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( A – C and E ) Unpaired t test. ( G , I , K , and L ) Unpaired t test with Bonferroni’s multiple-comparison adjustment.

Article Snippet: Cells were incubated in a 37°C CO 2 incubator for 24 hours before treatment with escalating concentrations of 17a (Cayman Chemical 17119) or carmofur (Selleck Chemicals S1289) for 72 hours.

Techniques: MTT Assay, Transfection, Control, Knockdown, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry, Infection, shRNA, Plasmid Preparation, Transduction, Exclusion Assay, Comparison

Journal: JCI Insight

Article Title: Upregulation of acid ceramidase contributes to tumor progression in tuberous sclerosis complex

doi: 10.1172/jci.insight.166850

Figure Lengend Snippet: ( A ) Female NSG mice were inoculated subcutaneously with 2 × 10 6 ELT3-luciferase–expressing cells. Weekly bioluminescence imaging was performed. Upon ELT3 tumor onset, mice were randomized and treated with vehicle or 17a (10 mg/kg/day, i.p.) for 4 weeks. ( B ) Tumor photon flux was quantified and normalized to the baseline measurements (week 0). Weekly bioluminescence imaging was performed. ( C ) 621-101 cells expressing luciferase (621L9) were infected with lentivirus of ASAH1 shRNA cells or control pLKO.1 empty vector. ASAH1 knockdown efficiency was determined by immunoblotting. β-Actin was used as a loading control. ( D ) Female NSG mice were subcutaneously inoculated with 2 × 10 6 621-101-pLKO.1 or 621-101-shASAH1 cells. Tumor formation was detected 21 weeks after cell inoculation. Weekly bioluminescence imaging was performed for up to 32 weeks. ( E ) Kaplan-Meier tumor-free survival curve. ( F ) Tumor photon flux was quantified and normalized to the baseline measurements (week 21) ( n = 3–4/group). ( G ) Female NSG mice were treated with vehicle or 17a (10 mg/kg/d, i.p.) for 2 days and then intravenously inoculated with 2 × 10 5 621L9 cells. Bioluminescence imaging was performed 1–24 hours after cell inoculation ( n = 3). ( H ) Photon flux at the chest region was quantified. ( I ) Female NSG mice were intravenously inoculated with 2 × 10 5 621L9-ASAH1-shRNA (#1 and #2) cells or control pLKO.1 cells. Bioluminescence imaging was performed 1–24 hours after cell inoculation ( n = 4–5). ( J ) Photon flux at the chest region was quantified. ( B , E , F , H , and J ) * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B , F , and H ) Unpaired t test. ( E ) Log-rank (Mantel-Cox) test. ( J ) Two-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Cells were incubated in a 37°C CO 2 incubator for 24 hours before treatment with escalating concentrations of 17a (Cayman Chemical 17119) or carmofur (Selleck Chemicals S1289) for 72 hours.

Techniques: Luciferase, Expressing, Imaging, Infection, shRNA, Control, Plasmid Preparation, Knockdown, Western Blot, Comparison

Journal: JCI Insight

Article Title: Upregulation of acid ceramidase contributes to tumor progression in tuberous sclerosis complex

doi: 10.1172/jci.insight.166850

Figure Lengend Snippet: Tsc2 +/– A/J mice were given vehicle, rapamycin, 17a, or rapamycin and 17a combinatorial treatment for 12 weeks ( n = 4 mice/group). Macroscopic analysis ( A ) and quantification ( B ) of renal tumor burden under a dissection microscope upon drug withdrawal. ( C ) Tumor rebound study and MRI follow-up posttreatment schema. ( D – F ) MRI of Tsc2 +/– A/J mouse kidneys at 4 and 8 weeks after withdrawal from treatment. ( G ) Mouse kidney sections were stained with H&E, p-S6 (Ser235/236), PCNA, and TUNEL. Scale bars are 20 μm except for the idicated 50 μm scale bar. ( H ) Percentages of cells with nuclear immunoreactivity for PCNA were scored from 5 random fields per section. * P < 0.05, ** P < 0.01, Mann-Whitney test. ( B , F , and H ) ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) Unpaired t test with Bonferroni’s multiple-comparison adjustment. ( F and H ) One-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Cells were incubated in a 37°C CO 2 incubator for 24 hours before treatment with escalating concentrations of 17a (Cayman Chemical 17119) or carmofur (Selleck Chemicals S1289) for 72 hours.

Techniques: Dissection, Microscopy, Staining, TUNEL Assay, MANN-WHITNEY, Comparison