cre recombinase adenovirus  (Vector Biolabs)

Journal: Cell reports

Article Title: Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation

doi: 10.1016/j.celrep.2023.112578

Figure Lengend Snippet: (A) Transmission electron microscopy (TEM) images of IDH non-mutant chondrosarcoma cells (n = 4) in vitro display absent glycogen granules. Organelles distinct from glycogen granules shown in magnified insets labeled as follows: M, mitochondria; L, lysosome; ER, rough endoplasmic reticulum. TEM images: 7× magnification, inset images: 70× magnification. (B) TEM images of mutant IDH chondrosarcoma cells (n = 7) display glycogen pools, asterisks denote aggregates of glycogen pools in mutant IDH cells, and arrows in magnified insets indicate glycogen pools. Glycogen appears as closely packed circular granules in mutant IDH chondrosarcoma patient cells. Images: 7× magnification, inset images: 70× magnification. (C) Glycogen quantification from pulverized patient-derived xenograft chondrosarcoma tissues display an elevation of glycogen in mutant IDH1 (n = 17) and IDH2 (n = 8) tumors compared with non-mutant tumors (n = 8). One-way ANOVA confirms significant statistical difference of glycogen levels between tumor genotypes (F(2,30) = 6.150, p = 0.0058). Tukey’s multiple comparisons test indicates that the mean values of glycogen in mutant IDH1 (p = 0.0107) and mutant IDH2 groups (p = 0.0109) were significantly higher than in non-mutant tumors. (D) PAS staining identified glycogen deposits in mutant IDH1 (n = 4) and IDH2 (n = 4) patient chondrosarcomas compared with non-mutant tumors (n = 5). PAS-D staining displays dissolution of glycogen deposits in mutant IDH tumors, thus confirming the presence of glycogen deposits. Arrows in magnified insets indicate glycogen deposits in cytoplasm of cells. Images: 40× magnification, inset images: 60× magnification. (E) Quantification of PAS-stained area (μm 2 ), normalized to total number of cells, and of PAS-D-stained area shows an elevation of glycogen deposits in mutant IDH1 (n = 4) and IDH2 (n = 4) chondrosarcomas compared with non-mutant tumors (n = 5). One-way ANOVA confirms significant statistical difference of glycogen levels between tumor genotypes (F(2,10) = 7.537, p = 0.0101). Tukey’s multiple comparisons test indicates mean values of glycogen in mutant IDH1 (p = 0.0380) and mutant IDH2 groups (p = 0.0123) were significantly higher than non-mutant tumors. (F) Glycogen deposits in Col2a1Cre; Idh1 LSL/wt E18.5 growth plates (n = 9) shown by PAS staining. Arrows in magnified insets indicate glycogen in cell cytoplasm. Glycogen deposits minimally in Col2a1Cre; Idh1 wt/wt control growth plates (n = 8). (G) Quantification of PAS-stained area (μm 2 ), normalized to total number of cells, and PAS-D-stained area (n = 8). (H) GYS1 staining is elevated in Col2a1Cre; Idh1 LSL/wt growth plates compared with Col2a1Cre; Idh1 wt/wt growth plates (n = 5). (I) Quantification of GYS1 staining from hypertrophic to resting zones (n = 5). (J) PYGL staining is unchanged in Col2a1Cre; Idh1 LSL/wt and Col2a1Cre; Idh1 wt/wt growth plates (n = 6). (K) Quantification of PYGL staining from hypertrophic to resting zones (n = 6). (L) Gene expression levels of glycogen genes are elevated upon Idh1 mutation induced by adenovirus Cre recombinase transfection (n = 3). Relative gene expression compared with adenovirus GFP control group was calculated and normalized to β-actin using the 2 −ΔΔCt method. One-way ANOVA with Tukey’s multiple comparisons test = p < 0.05. p value = Student’s t test p < 0.05, an asterisk (*) indicates that significant p values are shown. Means and error bars representing standard deviations are shown. Scale bars: 2 μm in white, 100 μm in black, and 10 μm in blue. Magnification: whole growth plate images: 13× magnification, inset images: 60× magnification.

Article Snippet: Ad-CMV-Cre Cre recombinase adenovirus co-expression with GFP , Vector Biolabs , 1700.

Techniques: Transmission Assay, Electron Microscopy, Mutagenesis, In Vitro, Labeling, Derivative Assay, Staining, Dissolution, Control, Gene Expression, Transfection

Journal: Cell reports

Article Title: Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation

doi: 10.1016/j.celrep.2023.112578

Figure Lengend Snippet: (A) HIF1⍺ staining is elevated in Col2a1Cre; Idh1 LSL/wt E18.5 growth plates compared with Col2a1Cre; Idh1 wt/wt growth plates (n = 3). Inset images taken at 20× magnification at the proliferative zone of the growth plate. (B) Quantification of HIF1⍺ staining from hypertrophic to resting zones (n = 3). (C) GLUT1 staining is elevated in Col2a1Cre; Idh1 LSL/wt growth plates compared with Col2a1Cre; Idh1 wt/wt growth plates (n = 3). Inset images taken at 20× magnification at the proliferative zone of the growth plate. (D) Quantification of GLUT1 staining from hypertrophic to resting zones (n = 3). (E) Gene expression levels of HIF1⍺ target genes are significantly reduced upon Idh1 mutation and HIF1⍺ knockdown induced by adenovirus Cre recombinase transfection (n = 3). (F) Gene expression levels of glycogen metabolism genes are significantly reduced upon Idh1 mutation and HIF1⍺ knockdown (n = 3). (G) Ex vivo explant cultures of E16.5 Col2a1Cre-ERT; Idh1 LSL/wt metatarsals display reduced glycogen (PAS) and HIF1⍺ levels upon pharmacological blockade of HIF1⍺ by 0.5 μM digoxin treatment for 4 days (n = 6). Inset images are taken at 15× magnification at the central area of the growth plate. p value = Student’s t test p < 0.05, an asterisk (*) indicates that significant p values are shown. Means and error bars representing standard deviations are shown. Scale bars: 100 μm in black, 50 μm in blue, and 20 μm in white.

Article Snippet: Ad-CMV-Cre Cre recombinase adenovirus co-expression with GFP , Vector Biolabs , 1700.

Techniques: Staining, Gene Expression, Mutagenesis, Knockdown, Transfection, Ex Vivo

Journal: bioRxiv

Article Title: APOBEC3 activity promotes the survival and evolution of drug-tolerant persister cells during acquired resistance to EGFR inhibitors in lung cancer

doi: 10.1101/2023.07.02.547443

Figure Lengend Snippet: A. qRT-PCR analysis showing A3A and A3B expression in PC9 cells following treatment with gefitinib or osimertinib over the course of 14 days. Error bars represent SEM of three technical replicates. B. Schematic of Cre-inducible APOBEC3B expression. C. qRT-PCR analysis showing A3B expression in PC9 cells following infection with Cre recombinase. Error bars represent SEM of three technical replicates. * indicates p<0.05. BT474 and SKBR3 cells are shown as controls. D. Western blot showing protein expression of HA-tagged A3B in PC9 cells following infection with Cre recombinase. E. In vitro deaminase activity assay in PC9 cells following infection with Cre recombinase. BT474 and SKBR3 cells are shown as controls. % deamination was calculated as described in Methods. F. Quantification of % deamination in two replicates of control PC9 cells (-Cre) and five replicates of A3B-expressing PC9 (+Cre). % deamination was calculated as described in Methods. Unpaired t-test was performed to determine statistical significance. ** indicates p<0.005. G. Growth curves for PC9 cells expressing A3B (+Cre) or control cells (-Cre). Error bars represent SEM of two biological replicates. Two-way ANOVA was performed to determine statistical significance. ns = not significant

Article Snippet: 24 hr later, adenovirus expressing Cre recombinase (Vector Biolabs) was added to the media at an MOI of 1000.

Techniques: Quantitative RT-PCR, Expressing, Infection, Western Blot, In Vitro, Activity Assay, Control