Journal: Scientific Reports

Article Title: Autofluorescence is a Reliable in vitro Marker of Cellular Senescence in Human Mesenchymal Stromal Cells

doi: 10.1038/s41598-019-38546-2

Figure Lengend Snippet: Western blot analysis of lipofuscin-related proteins. The protein expression of GGH, FAM96B and HDGFL1 was compared in low (early passages) and high (late passages) autofluorescent MSC. ( a ) Data was quantified and normalized to β-actin expression. ( b ) Results are represented as fold changes compared to the respective low autofluorescence MSC (red dotted line). (n = 3; values represent the mean ± SEM).

Article Snippet: The nitrocellulose membranes were incubated with mouse monoclonal antibodies against γ-glutamyl hydrolase (GGH; 1:250; rabbit, HPA025226, Sigma), family with sequence similarity 96, member B (FAM96B; 1:250; rabbit, HPA041736, Sigma), hepatoma derived growth factor-like-1 (HDGFL1; 1:250; rabbit, HPA045679, Sigma), and housekeeping gene β-actin (1:5000; mouse, HRP conjugate, 12262, Cell Signaling – Bio Concept).

Techniques: Western Blot, Expressing

Journal: Cell metabolism

Article Title: Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells.

doi: 10.1016/j.cmet.2020.10.017

Figure Lengend Snippet: Figure 1. Metabolism-Focused CRISPR Screens In Vivo Reveal Metabolic Dependencies of Pancreatic Tumors (A) Schematic of genetics screens to identify metabolic dependencies of KP pancreatic cancer specifically in vivo. (B) Cumulative frequency curve of represented guides in genetic screens. (C) Gene scores of in vivo versus in vitro genetic screens of KP pancreatic cancer growth. (D) Volcano plot of differential gene scores comparing in vivo against in vitro conditions (left). Top 20 genes scoring as differentially required in vivo. Genes involved in specific metabolic pathways are indicated (right). (E) Gene sets enriched in differentially required genes in vivo versus in vitro for pancreatic cancer growth. The heatmap generated by iPAGE represents the extent to which each gene set is enriched among the genes that are essential for tumor growth in vivo. See also Figure S1.

Article Snippet: CRISPR Cas9 sgRNA KO mouse metabolism library generated in this study has been deposited to Addgene (ID 160129).

Techniques: CRISPR, In Vivo, In Vitro, Generated

Journal: Cell metabolism

Article Title: Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells.

doi: 10.1016/j.cmet.2020.10.017

Figure Lengend Snippet: Figure 3. Heme Synthesis Is a Metabolic Dependency of Kras-Driven Tumors In Vivo (A) Immunoblot of HMBS in the indicated KP pancreas and KP lung cancer cell lines. GAPDH was used as loading control. (B) Fold change in cell number (log2) of the indicated KP pancreas and KP lung cancer cell lines after culturing in vitro for the indicated durations (mean ± SD, n = 3). ***p < 0.001 versus sgControl. (C) Tumor weights of the indicated KP pancreas and KP lung tumors engrafted subcutaneously in C57BL/6J mice (box and whisker, n = 8). *p < 0.05, ***p < 0.001 versus sgControl (left). Images of the indicated KP pancreas and KP lung tumors (right). (D) Immunoblot of HMOX1 in KP pancreas and KP lung cancer cells grown in vitro under normoxia, under hypoxia (0.5% oxygen) for 48 h, and in subcutaneous tumors. GAPDH was used as loading control. (E) Immunoblots of HMOX1 and HMBS in the indicated KP pancreas cell lines. GAPDH was used as loading control. (F) Relative tumor weights of the indicated KP pancreas Hmbs_KO tumors engrafted subcutaneously in C57BL/6J mice (box and whisker, n = 23). *p < 0.05 versus control (top). Representative image of the indicated KP pancreas Hmbs_KO tumors (bottom). (G) Schematic of competition assay using PDAC patient-derived xenograft cells infected with the indicated sgRNAs. Cells were then engrafted subcutaneously in NSG mice (left). Relative fold change in sgRNA abundance (log2) from the PDX (mean ± SD, n = 5). **p < 0.01 versus sgControl (right). (H) Disease-free survival rates of TCGA PDAC patients with high or low heme synthesis gene expressions. Weighted average expressions of CPOX, HMBS, PPOX, and UROS were used (low heme n = 83, high heme n = 28). See also Figure S2.

Article Snippet: CRISPR Cas9 sgRNA KO mouse metabolism library generated in this study has been deposited to Addgene (ID 160129).

Techniques: In Vivo, Western Blot, Control, In Vitro, Whisker Assay, Competitive Binding Assay, Derivative Assay, Infection

Journal: Scientific Reports

Article Title: Autofluorescence-based sorting removes senescent cells from mesenchymal stromal cell cultures

doi: 10.1038/s41598-020-76202-2

Figure Lengend Snippet: RNA and protein expression of MSC-related senescence markers. The expression of senescence markers in MSC (n = 3) was analysed in unsorted and sorted groups—LA, MA and HA—and compared to control. Gene expression of positive—p16 INK4A ( a ), p21 CIP1 ( b ) and ANKRD1 ( c )—and negative— p18 INK4C ( d ) CDCA7 ( e ) and E2F1 ( f )—senescence markers was tested by qPCR (normalized to beta-actin and PPIA). The protein expression of p21 CIP1 , p53, Retinoblastoma (Rb) and p16 INK4A was compared between control, unsorted and sorted MSC groups by semi-quantitative Western blot ( g ).

Article Snippet: The nitrocellulose membranes were incubated with mouse monoclonal antibodies against anti-p53 (mouse, OP43, Calbiochem, Sigma), anti-p21 (rabbit, 2947, Cell Signaling Technology—Bio Concept), anti-p16 (mouse, NA29, Calbiochem), anti-RB (mouse, OP66, Calbiochem) and housekeeping gene β-actin (mouse, HRP conjugate, 12,262, Cell Signaling Technology).

Techniques: Expressing, Control, Gene Expression, Western Blot