Journal: The EMBO Journal

Article Title: Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis

doi: 10.15252/embj.2020105111

Figure Lengend Snippet: Western analysis for on‐target effects for everolimus (EV, P‐RPS6) and CX‐5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX‐5461 vehicle: 25 mM NaH 2 PO 4 ; V/V; mouse #1–6), 5 mg/kg everolimus (EV; mouse #7–12), 35 mg/kg CX‐5461 (mouse #13–18), or both drugs (CX‐5461 + EV; mouse #19–24) for 2 h ( n = 6 per treatment group). Actin was used as a loading control. A schematic illustration of the polysome profiling analysis workflow: Cytoplasmic lysate was layered on top of a linear 10–40% sucrose gradient, ultracentrifuged (222,228 g , 2¼ h at 4°C using SW41Ti rotor), and fractionated using the Foxy Jr Fraction Collector with constant monitoring of absorbance at 260 (A260) nm by an ISCO UA‐6 Absorbance Detector. Fractions (one fraction per minute: 800 μl per tube) corresponding to polysomal mRNAs that were bound by four or more ribosomes were pooled and analyzed by RNA‐seq followed by data analysis using anota2seq or limma. Enrichment analysis by MetaCore ® GeneGO of genes in “translation up” (red) and “translation down” (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX‐5461 + EV treatment group with the V/V group ( n = 6). “Ratio” values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore ® 's database. Genes implicated in “ Translation: initiation ” and “ Translation: Elongation‐Termination ” processes based on Fig C and Fig E. log 2 FC: log 2 fold change; FDR: false discovery rate (adjusted P value). Activity levels of key biological processes involved in cellular growth, proliferation, and metabolism based on single sample gene set enrichment analysis (ssGSEA) of indicated comparisons. Percentage increase (red) or decrease (blue) in the activity levels of key biological processes involved in cellular growth, proliferation, and metabolism based on ssGSEA of indicated comparisons. Data were obtained from n = 6 mice per treatment group. Source data are available online for this figure.

Article Snippet: The cytoplasmic lysate was layered on top of a linear 10–40% sucrose gradient, ultracentrifuged (SW41 rotor, 222,228 g , 2¼ h at 4°C using SW41Ti rotor), and fractionated using the Foxy Jr Fraction Collector with constant monitoring of absorbance at 260 nm by an ISCO UA‐6 Absorbance Detector (Teledyne).

Techniques: Western Blot, Isolation, RNA Sequencing Assay, Activity Assay

Journal: Scientific Reports

Article Title: Intra-striatal AAV2.retro administration leads to extensive retrograde transport in the rhesus macaque brain: implications for disease modeling and therapeutic development

doi: 10.1038/s41598-020-63559-7

Figure Lengend Snippet: AAV2.retro-mediated retrograde transport is significantly higher compared to the parent serotype, AAV2. ( a ) Low and ( b ) high power photomicrographs of eGFP-stained, rhesus macaque coronal brain sections following injection of AAV2.retro-eGFP (top panel) or AAV2-eGFP (bottom panel) into the caudate and putamen. Rectangles in ( a ) illustrate the brain regions selected for high power photomicrographs displayed in (b ). ( c ) Cell count analysis demonstrating significantly more GFP + cells in the putamen compared to the caudate, irrespective of serotype, as well as significantly more GFP + cells detected in extra-striatal regions in animals injected with AAV2.retro-eGFP compared to AAV2-eGFP. ( d ) Area fraction fractionator analysis showing a significantly higher area fraction of GFP positivity in the putamen in animals injected with AAV2-eGFP compared to AAV2.retro-eGFP. Error bars in both graphs represent standard error of the mean (SEM). Abbreviations: ACC (anterior cingulate cortex), AMY (amygdala), Cd (caudate), DPFC (dorsal prefrontal cortex), DPMC (dorsal premotor cortex), Put (putamen), SSC (somatosensory cortex), THAL (thalamus). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 2a = 1 centimeter, Scale bar 2b = 100 microns. Graphs were made in GraphPad Prism for Mac, Version 8.

Article Snippet: The Area Fraction Fractionator (MBF Bioscience) was used to quantify the area fraction of eGFP + cells in the putamen and caudate.

Techniques: Staining, Injection, Cell Counting

Journal: Scientific Reports

Article Title: Intra-striatal AAV2.retro administration leads to extensive retrograde transport in the rhesus macaque brain: implications for disease modeling and therapeutic development

doi: 10.1038/s41598-020-63559-7

Figure Lengend Snippet: AAV2.retro-mediated retrograde transport is significantly higher compared to the parent serotype, AAV2. ( a ) Low and ( b ) high power photomicrographs of eGFP-stained, rhesus macaque coronal brain sections following injection of AAV2.retro-eGFP (top panel) or AAV2-eGFP (bottom panel) into the caudate and putamen. Rectangles in ( a ) illustrate the brain regions selected for high power photomicrographs displayed in (b ). ( c ) Cell count analysis demonstrating significantly more GFP + cells in the putamen compared to the caudate, irrespective of serotype, as well as significantly more GFP + cells detected in extra-striatal regions in animals injected with AAV2.retro-eGFP compared to AAV2-eGFP. ( d ) Area fraction fractionator analysis showing a significantly higher area fraction of GFP positivity in the putamen in animals injected with AAV2-eGFP compared to AAV2.retro-eGFP. Error bars in both graphs represent standard error of the mean (SEM). Abbreviations: ACC (anterior cingulate cortex), AMY (amygdala), Cd (caudate), DPFC (dorsal prefrontal cortex), DPMC (dorsal premotor cortex), Put (putamen), SSC (somatosensory cortex), THAL (thalamus). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 2a = 1 centimeter, Scale bar 2b = 100 microns. Graphs were made in GraphPad Prism for Mac, Version 8.

Article Snippet: To assess these serotype-based differences, we quantified the spread of each vector in the injected regions of the caudate and putamen using the Area Fraction Fractionator tool (MBF Bioscience), calculating the area of eGFP + cells per area of each structure.

Techniques: Staining, Injection, Cell Counting