Journal: Nucleic Acids Research

Article Title: A conserved nutrient responsive axis mediates autophagic degradation of miRNA–mRNA hybrids in blood cell progenitors

doi: 10.1093/nar/gkad1047

Figure Lengend Snippet: Targeting miRNAs and amino acid sensing in Drosophila blood cell progenitors. ( A–A' ) The primary lobe of the lymph gland was isolated from a 48 h AEH larvae by laser capture micro-dissection (LCM) technique. ( B ) Marker analysis was done after cDNA conversion of RNA isolates post-LCM dissection of the primary lobes, which is then visualized through Agarose gel electrophoresis. ( C ) Heatmap depicting the expression profile of the Drosophila miRNAs present in the primary lobe (48 h AEH) retrieved through LCM. ( D–F' ) EdU incorporation analysis of lymph glands at (D, D') 36 h, ( E, E' ) 48 h and ( F, F' ) 60 h upon Dicer1 down regulation from the progenitor. ( G–G' ) Volumetric comparison by 3D-reconstruction of progenitors in the wildtype ( G ) and ( G' ) Dcr1 knockdown lymph gland. (H–K) Quantitative time-kinetic analysis of Wildtype and Dcr1KD progenitors depicting the ( H ) proliferation rate, ( I ) progenitor number, ( J ) progenitor volume and ( K ) individual progenitor cell volume observed at 36, 48 and 60 h AEH respectively. ( L–N' ) Quantitative analysis of progenitor number and volume upon increasing concentration of Carbon sources. ( L–L' ) in excess Carbohydrates, ( M–M' ) in excess lipids, ( N–N' ) in excess proteins. Scale, 20 μm in all images. Individual dots represent ‘ n ’ of the sample. Significance was evaluated using two-way ANOVA with Tukey's test was performed for grouped analyses. Error bar: standard deviation (SD). Data are mean ± SD. * P < 0.033, ** P < 0.002 and *** P < 0.001. See also .

Article Snippet: Primary Bone Marrow CD34+ Hematopoietic Cells, Normal, Human were acquired from ATCC (PCS-800-012).

Techniques: Isolation, Dissection, Marker, Agarose Gel Electrophoresis, Expressing, Comparison, Knockdown, Concentration Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A conserved nutrient responsive axis mediates autophagic degradation of miRNA–mRNA hybrids in blood cell progenitors

doi: 10.1093/nar/gkad1047

Figure Lengend Snippet: Human blood progenitors exhibit leucine responsive proliferation similar to Drosophila . ( A–A' ) EdU incorporation analysis of CD34+ CD10-Lin- myeloid biased HSPC in undeprived and leucine-deficient media. ( B and B' ) Quantitative analysis in the proliferation rate of CD34+ CD10-Lin- HSPCs and KG1 cells. ( C–D' ) Dose-dependent recovery in the proliferation rate of CD34+ CD10-Lin– HSPC upon increasing concentration of leucine in leucine deprived media. ( D–D' ) Quantitative analysis of the recovery in proliferation index of CD34+ CD10-Lin- HSPCs and KG1 cells. ( E ) OPP incorporation rate analysis of CD34+ CD10-Lin-myeloid biased HSPC in early stages (6 h) of leucine deprivation versus late stages (16 h). ( F ) Lysotracker staining of acidic autolysosome upon leucine starvation at an early time point in HSPCs. ( G ) Cyto-ID staining of autophagosomes in response to leucine starvation at early time point in HSPCs. ( H ) Quantitative analysis of OPP incorporation rate at 6 and 16 h of leucine starvation condition in HSPCs. ( I ) Quantitative measurement of increased acidic vesicles (Lysotracker) in HSPCs. ( J ) Quantitative measurement of autophagosomes (Cyto-ID) in HSPCs. Scale, 20 μm in all images. Individual dots represent ‘n’ of the sample. Two-way ANOVA was performed for grouped analyses. One-way ANOVA was performed for individual multiple comparisons. Error bar: standard deviation (SD). Data are mean ± SD. * P < 0.033, ** P < 0.002 and *** P < 0.001. See also .

Article Snippet: Primary Bone Marrow CD34+ Hematopoietic Cells, Normal, Human were acquired from ATCC (PCS-800-012).

Techniques: Concentration Assay, Staining, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A conserved nutrient responsive axis mediates autophagic degradation of miRNA–mRNA hybrids in blood cell progenitors

doi: 10.1093/nar/gkad1047

Figure Lengend Snippet: Leucine-based proliferation of human blood progenitors also targets autophagy-dependent miRNA turnover. ( A ) Heatmap representing high throughput transcriptomic analysis at 6 h pulse of leucine starvation in CD34+ CD10-Lin– HPSCs. ( B ) Network map depicting pathway characterization of the differentially expressing genes in CD34+ CD10-Lin– HSPC as a response to leucine deprivation. ( B' ) GO analysis of the most significantly affected biological process in CD34+ CD10-Lin- HSPCs as a response to leucine deprivation. ( C ) Western blot depicting reduction in Ago2 protein upon early and late response to leucine starvation. ( D ) Levels of p62/SQSTM1 show a similar reduction as in (C). ( E ) Co-immunoprecipitation depicting physical interaction between p62 and Ago2. The blot depicts a basal level of Ago2 interaction that increases upon leucine deprivation (Low exposure of the same blot is in ). ( F ) EdU incorporation analysis upon leucine starvation and simultaneous treatment with BafilomycinA1 (BafA1). ( F' ) Quantitative analysis on the proliferation rate upon leucine deprivation and BafA1 treatment. ( G ) Levels of Ago-2 upon leucine deprivation and BafA1 treatment detected by western blotting. ( H ) Quantitative analysis of Ago2 protein levels upon leucine deprivation and BafA1 treatment. ( I ) Schematic representing miRNA-9 sponge construct used for RNA-IP (above). Destabilized GFP transcript with deleted sponge sites (served as Control-Sponge) and destabilized GFP transcript with 8x sponge sites (miRNA-9 Sponge) (below). ( J ) Agarose gel demonstrates a qualitative assessment of miRNA-9 sponge enrichment upon p62-RNA-IP. ( J' ) A qPCR-based analysis of Control-sponge and miRNA-9 sponge enrichment upon p62-RNA-IP and assessment of an endogenous transcript (actin) within the pulled fraction. ( K ) qPCR analysis of RNA-IP done with p62 as bait using Control-sponge and miRNA-9 sponge construct upon leucine deprivation (left). qPCR analysis of puromycin transcript acts as a negative control (right). ( K' ) qPCR analysis of RNA-IP done with p62 on miRNA-9 sponge construct upon leucine deprivation and BafA1 treatment during leucine deprivation (left). A qPCR analysis of GFP in the Input fraction of the same (right). Scale, 20 μm in all images. Individual dots represent ‘n’ of the sample. Multiple t -test with Welch correction was performed for RNA-IP analysis. One-way ANOVA was performed for individual multiple comparisons. Error bar: standard deviation (SD). Data are mean ± SD. * P < 0.033, ** P < 0.002 and *** P < 0.001.

Article Snippet: Primary Bone Marrow CD34+ Hematopoietic Cells, Normal, Human were acquired from ATCC (PCS-800-012).

Techniques: High Throughput Screening Assay, Expressing, Western Blot, Immunoprecipitation, Construct, Control, Agarose Gel Electrophoresis, Negative Control, Standard Deviation