Journal: International Journal of Molecular Sciences

Article Title: Cold Atmospheric Plasma Does Not Affect Stellate Cells Phenotype in Pancreatic Cancer Tissue in Ovo

doi: 10.3390/ijms23041954

Figure Lengend Snippet: CAP does not alter the activation profile of RLT-PSC cells in ovo. Sections of RLT-PSC and co-cultured RLT-PSC + Mia PaCa-2 from in ovo tissue were stained for GFAP, vimentin (Vim), and ACTA-2. ( a – c , g – i ) Mean fluorescence intensity (MFI) expressed as arbitrary units (a.u.); ( d – f , j – l ) number of cells positive for the corresponding markers. Anti-CD44 antibodies (PDAC cells) were used as a counterstain to discriminate Mia PaCa-2 cells from RLT-PSC. UT = untreated control. n ≥ 4 tissue per condition; each dot represents one tissue. Dashed lines = median; dotted lines = 25% and 75% quartiles; * = p ≤ 0.05.

Article Snippet: Primary antibodies used: GFAP (HPA056030, Atlas Antibodies, 1/200, Bromma, Switzerland); ACTA-2 (M0851, Dako, 1/200); Vimentin (Abcam ab92547, 1/500, Cambridge, UK); CD44 (3570S, Cell Signaling Technologies, 1/400, Danvers, MA, USA); MMP2 (40994, Cell Signaling Technologies, 1/400, Danvers, MA, USA); MMP9 (13667, Cell Signaling Technologies, 1/100, Danvers, MA, USA).

Techniques: Activation Assay, In Ovo, Cell Culture, Staining, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Cold Atmospheric Plasma Does Not Affect Stellate Cells Phenotype in Pancreatic Cancer Tissue in Ovo

doi: 10.3390/ijms23041954

Figure Lengend Snippet: List of primers used for quantitative real-time PCR.

Article Snippet: Primary antibodies used: GFAP (HPA056030, Atlas Antibodies, 1/200, Bromma, Switzerland); ACTA-2 (M0851, Dako, 1/200); Vimentin (Abcam ab92547, 1/500, Cambridge, UK); CD44 (3570S, Cell Signaling Technologies, 1/400, Danvers, MA, USA); MMP2 (40994, Cell Signaling Technologies, 1/400, Danvers, MA, USA); MMP9 (13667, Cell Signaling Technologies, 1/100, Danvers, MA, USA).

Techniques:

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) Gene expression profiling from SOX2-eGFP + and eGFP − sorted cells isolated from tumors developed in eGFP-Sox2; Ptch1 +/− mice (RNA-seq data obtained from Dirks, 2014 dataset) was analyzed using a Kyoto Encyclopedia of Genes and Genomes (KEGG) Hedgehog signaling signature. A heatmap comparing gene expression is shown. Arrowhead indicates differentially expressed gene ( P = 0.014). ( B ) Spontaneous MB tissues developed in Ptch1-Gfap or ND2:SmoA1 mice, two independent PDOX-derived tissues [SJSHHMB-14-7196 (SJSHHMB) and RCMB18], or a human MB TMA were stained for GLI2 and SOX2. ( C ) Expression of Sox2 in a t-SNE projection of cells was visualized by analyzing previously run scRNA-seq data from spontaneous SmoM2 MB (sequencing data from Ocasio, 2019 dataset). ( D ) A similar scRNA-seq dataset was used to show expression of SHH biomarkers across indicated nodes. ( E ) Spontaneous MB developed in Ptch1-Gfap mice, from mice harboring an SHH subgroup PDOX (RCMB18) or from a TMA were stained for SOX2 and GFAP. ( F ) Percentages of Sox2 + cells from SmoM2 tumors treated with vehicle or vismodegib in the indicated cell clusters were obtained using Ocasio’s transcriptomic data. The numbers of Sox2 + cells were normalized to total sample cell count. Data shown represent the average percentage for each sequenced tumor. ( G ) Percentages of Gli1/Gli2 + cells in vehicle or vismodegib-treated SmoM2 tumors were similarly calculated. ( H ) A density blot showing Gli1 / Gli2 / Sox2 expression in a t-SNE projection of cells from similarly treated tumors. Square details density on astrocytic cluster. Representative IHCs, with highlighted double-labeled cells, are shown. Scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: For flow cytometry analyses, cell suspensions were stained using a LIVE/DEAD viability kit (Invitrogen) and then incubated in Cytofix/Cytoperm solution (Becton Dickinson) before staining using corresponding fluorescent-conjugated antibody: SOX2-APC (BioLegend), GLI1-APC-Cy7, GLI2-FITC, and GFAP-PE (Novus Biologicals).

Techniques: Expressing, Isolation, RNA Sequencing Assay, Derivative Assay, Staining, Sequencing, Cell Counting, Labeling

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) Indicated proteins were stained in MPC1 cultures, and percentages of positive cells were determined by flow cytometry analyses. ( B ) MPC2 cultures were exposed to cyclopamine (10 μM) or its inactive analog tomatidine (10 μM), vismodegib (100 nM), or dimethyl sulfoxide (DMSO) for 16 hours before staining for bromodeoxyuridine (BrdU) and cleaved caspase-3. ( C ) MPC2 cultures were exposed to GANT-61 for 16 hours, and expression of indicated genes was determined. ( D ) Indicated cultures were exposed to GANT-61 for 72 hours before determining cell viability by MTT [3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2 H- tetrazolium bromide] reduction. ( E ) MPC1 cultures were exposed to I-BET151 for 16 hours before determining the expression of indicated genes. ( F ) MPC cultures were exposed to I-BET151 for 72 hours before determining cell viability by MTT reduction. ( G ) MPC1 cultures were exposed to I-BET151 (500 nM) for 16 hours before assay BrdU incorporation and cleaved caspase-3 expression. ( H ) MPC1 cultures were exposed to I-BET151 (500 nM) for 24 hours, and percentages of positive cells were determined by flow cytometry. ( I ) Percentage of GFAP + cells in a SOX2/GLI2 + pool was similarly determined. ( J ) MPC2 cultures were transfected with pooled Genome siRNA sequences targeting the indicated genes or a scramble siRNA control ( siSC ), and expression of SHH target genes was determined 72 hours later. ( K ) MPC2 cultures were transfected with similar siRNAs or a GFP -labeled siRNA. Cell viability was determined 5 days later by MTT reduction. ( L ) MPC1 cells were similarly transfected, and numbers of SOX2 + cells were determined by flow cytometry. ( M ) MPC1 cultures were transfected with indicated vectors and, 48 hours later, exposed to I-BET151 (500 nM) for additional 72 hours. Cell viability was determined by MTT reduction. ( N ) MPC1 cultures were transfected with indicated Genome siRNA sequences. Forty-eight hours after transfection, cells were exposed to I-BET151 (500 nM) for additional 72 hours, and cell viability was similarly determined. Scale bars, 100 μm. * P ≤ 0.05.

Article Snippet: For flow cytometry analyses, cell suspensions were stained using a LIVE/DEAD viability kit (Invitrogen) and then incubated in Cytofix/Cytoperm solution (Becton Dickinson) before staining using corresponding fluorescent-conjugated antibody: SOX2-APC (BioLegend), GLI1-APC-Cy7, GLI2-FITC, and GFAP-PE (Novus Biologicals).

Techniques: Staining, Flow Cytometry, Expressing, BrdU Incorporation Assay, Transfection, Control, Labeling

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) MPC cultures were exposed to JQ-1 for 72 hours before determining cell viability by MTT reduction. ( B ) MPC47 cells were exposed to JQ-1, and expression of indicated genes was determined 16 hours later. ( C ) Mice harboring Ptch1-LacZ (MB47) subcutaneous tumors were treated with JQ-1 (30 mg/kg, i.p., q.d.) or vehicle for 8 days, before determining levels of the indicated proteins. ( D ) Expression of indicated genes was determined in similarly dosed mice. ( E ) Mice harboring similar tumors were treated with JQ-1 (30 mg/kg, i.p., q.d.), vismodegib (25 mg/kg, i.p., q.d.), or vehicle for 8 days, and the tumor volume was measured. ( F ) Mice harboring orthotopic MB47 tumors were similarly dosed, and tumor area was quantified. Scale bars, 200 μm. ( G ) Number of SOX2 + cells in brain tumors from similarly treated mice were quantified. ( H ) Mice harboring subcutaneous MB47 tumors were treated with vehicle or JQ-1 (30 mg/kg, i.p., q.d.) for 8 days, before determining percentage of positive cells by flow cytometry. ( I ) Percentages of GFAP + in SOX2/GLI2 + cells were similarly determined. ( J ) Mice harboring subcutaneous MB47 were treated with JQ-1 (30 mg/kg, i.p., q.d.), vismodegib (25 mg/kg, i.p., q.d.), or vehicle for 8 days. Equal numbers of viable cells from residual tumors were then allowed to form spheres ex vivo or to orthotopically engraft in vivo. A schematic of the procedure is shown. ( K ) Numbers of spheres grown from treated tumors described in (J) were quantified. ( L ) Tumor engraftment capability from similar residual tumors was determined. ( M ) PDOX harboring mice were dosed with vehicle, vismodegib (25 mg/kg, i.p., q.d.), or JQ-1 (30 mg/kg, i.p., q.d.) for 8 days, before staining brains for SOX2. Unless otherwise indicated, scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: For flow cytometry analyses, cell suspensions were stained using a LIVE/DEAD viability kit (Invitrogen) and then incubated in Cytofix/Cytoperm solution (Becton Dickinson) before staining using corresponding fluorescent-conjugated antibody: SOX2-APC (BioLegend), GLI1-APC-Cy7, GLI2-FITC, and GFAP-PE (Novus Biologicals).

Techniques: Expressing, Flow Cytometry, Ex Vivo, In Vivo, Staining

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) Light2 cells were exposed to 100 nM SAG for 24 h, before exposing them to BMS-986158. GLI- driven luciferase activity was determined 16 hours later. ( B ) WT MEFs were similarly treated, before immunoblot protein lysates for indicated proteins. ( C ) Expression of the indicated genes in similarly treated MEFs was determined. ( D ) Sufu −/− MEFs were treated with BMS-986158 for 16 hours, and Gli1 expression was determined. ( E ) WT MEFs were treated with SAG (100 nM) for 24 hours before their exposure to BMS-986158 for additional 16 hours. BRD4 occupancy in Gli1 locus was analyzed by ChIP-PCR and normalized to a ChIP performed using rabbit IgG. Primer set 3 (PS3) was used as a negative control, while PS6 to PS8 aligned with Gli1 promoter. ( F ) GCPs were induced with 100 nM SAG for 24 hours and exposed to BMS-986158 for additional 16 hours, before assay BrdU incorporation and stain-cleaved caspase-3. Scale bar, 100 μm. ( G ) Vismodegib-sensitive SHH-47 cells were exposed to BMS-986158 for 16 hours, and expression of indicated proteins was determined. ( H ) Expression of indicated genes was assayed in similarly treated cells. ( I ) Indicated cells were exposed to BMS-986158 for 72 hours, and cell viability was determined by MTT reduction. ( J ) Mice harboring subcutaneous MB47 ( Ptch1-LacZ ) and MB1 ( Ptch1-LacZ , Trp53-KO ) tumors were dosed with BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle for 8 days, before determining levels of indicated proteins. ( K ) Tumor size in similarly treated mice was determined. ( L ) Ten-day-old Ptch1-Gfap mice were dosed for 8 days with vehicle or BMS-986158 (3 mg/kg, i.p., q.d.), and the size of tumors was quantified. Scale bar, 200 μm. ( M ) Brains from similarly treated Ptch1-Gfap mice were stained for the indicated proteins. Scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: For flow cytometry analyses, cell suspensions were stained using a LIVE/DEAD viability kit (Invitrogen) and then incubated in Cytofix/Cytoperm solution (Becton Dickinson) before staining using corresponding fluorescent-conjugated antibody: SOX2-APC (BioLegend), GLI1-APC-Cy7, GLI2-FITC, and GFAP-PE (Novus Biologicals).

Techniques: Luciferase, Activity Assay, Western Blot, Expressing, Negative Control, BrdU Incorporation Assay, Staining

Journal: Science Advances

Article Title: Noncanonical activation of GLI signaling in SOX2 + cells drives medulloblastoma relapse

doi: 10.1126/sciadv.abj9138

Figure Lengend Snippet: ( A ) MPC47 cultures were exposed to BMS-986158 for 16 hours before determining expression of SHH target genes. ( B ) MPC1 cultures were similarly exposed to BMS-986158, and expression of indicated genes was determined. ( C ) MPC cultures were exposed to BMS-986158 for 72 hours before determining cell viability by MTT reduction. ( D ) Mice harboring subcutaneous MB47 ( Ptch1-LacZ ) and MB1 ( Ptch1-LacZ , Trp53-KO ) tumors were exposed to BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle for 8 days, and levels of indicated proteins were determined. ( E ) Ten-day-old Ptch1-Gfap mice were treated with vehicle or BMS-986158 (3 mg/kg, i.p., q.d.) for 8 days, and SOX2 was detected by IHC staining. ( F ) Mice harboring subcutaneous MB47 were treated with BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle for 8 days. As shown in , equal number of viable cells from residual tumors were then allowed to form spheres ex vivo. ( G ) As represented in tumor engraftment capability from similarly treated tumors was determined. ( H ) Ten-day-old Ptch1-Gfap mice were treated with BMS-986158 (3 mg/kg, i.p., q.d.) or vehicle for 8 days before labeling brain tissues with indicated metal conjugated antibodies. Representative multiplexing mass cytometry images and corresponding H&E staining are shown. Scale bars, 200 μm. ( I ) Ptch1-Gfap mice were similarly treated before staining brains for SOX2 and GFAP. Unless otherwise indicated, scale bars, 50 μm. * P ≤ 0.05.

Article Snippet: For flow cytometry analyses, cell suspensions were stained using a LIVE/DEAD viability kit (Invitrogen) and then incubated in Cytofix/Cytoperm solution (Becton Dickinson) before staining using corresponding fluorescent-conjugated antibody: SOX2-APC (BioLegend), GLI1-APC-Cy7, GLI2-FITC, and GFAP-PE (Novus Biologicals).

Techniques: Expressing, Immunohistochemistry, Ex Vivo, Labeling, Multiplexing, Mass Cytometry, Staining