Journal: Molecular medicine reports

Article Title: Long non‑coding RNA SNHG14 affects the proliferation and apoptosis of childhood acute myeloid leukaemia cells by modulating the miR‑193b‑3p/MCL1 axis.

doi: 10.3892/mmr.2020.11729

Figure Lengend Snippet: Figure 1. SNHG14 gene expression is upregulated in bone marrow tissues of patients with AML and AML cell lines. (A) Relative expression of SNHG14 in 57 AML bone marrow tissues and NBM. (B) Relative expression of SNHG14 in AML cell lines and human normal bone marrow CD34+ cells. **P<0.01 vs. CD34+ cells. The 2‑ΔΔCq method was used to analyse the relative mRNA expression level using GAPDH as an internal control. AML, acute myeloid leukaemia; NBM, normal marrow tissues; SNHG14, small nucleolar RNA host gene 14.

Article Snippet: Human normal bone marrow CD34+ cells and AML cell lines (MV‐4‐11, AML‐193, HL‐60, and KG‐1 cells) were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C.

Techniques: Gene Expression, Expressing, Control

Journal: Molecular medicine reports

Article Title: Long non‑coding RNA SNHG14 affects the proliferation and apoptosis of childhood acute myeloid leukaemia cells by modulating the miR‑193b‑3p/MCL1 axis.

doi: 10.3892/mmr.2020.11729

Figure Lengend Snippet: Figure 3. miR‑193b‑3p is a target of SNHG14 in AML cells. (A) Starbase was used to predict the binding site between SNHG14 and miR‑193b‑3p. (B) Relative expression of miR‑193b‑3p in MV‑4‑11 and AML‑193 cells following SNHG14 silencing. **P<0.01 vs. blank control. (C) The target association between SNHG14 and miR‑193b‑3p was determined using an RNA immunoprecipitation assay. **P<0.01 vs. Anti‑IgG. (D) The target association between SNHG14 and miR‑193b‑3p was determined using a dual luciferase reporter gene assay. **P<0.01 vs. miR‑NC. (E) Relative expression of miR‑193b‑3p in 57 AML bone marrow tissues and NBM was detected by RT‑qPCR. (F) Spearman's correlation analysis was performed to evaluate the correlation between SNHG14 and miR‑193b‑3p expression. (G) Relative expression of miR‑193b‑3p in AML cell lines and human normal bone marrow CD34+ cells. **P<0.01 vs. CD34+ cells. The 2‑ΔΔCq method was used to analyse the relative mRNA expression level using U6 as an internal control. SNHG14, small nucleolar RNA host gene 14; miR, microRNA; si, small interfering RNA; NC, negative control; wt, wildtype; mut, mutated; NBM, normal marrow tissues; AGO2, protein argonaute‑2; AML, acute myeloid leukaemia.

Article Snippet: Human normal bone marrow CD34+ cells and AML cell lines (MV‐4‐11, AML‐193, HL‐60, and KG‐1 cells) were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C.

Techniques: Binding Assay, Expressing, Control, RNA Immunoprecipitation, Luciferase, Reporter Gene Assay, Small Interfering RNA, Negative Control

Journal: Molecular medicine reports

Article Title: Long non‑coding RNA SNHG14 affects the proliferation and apoptosis of childhood acute myeloid leukaemia cells by modulating the miR‑193b‑3p/MCL1 axis.

doi: 10.3892/mmr.2020.11729

Figure Lengend Snippet: Figure 5. miR‑193b‑3p targets MCL1 in AML cells. (A) TargetScan was used to predict the binding site between miR‑193b‑3p and MCL1. (B) A dual luciferase reporter gene assay was employed to verify the target association between miR‑193b‑3p and MCL1. **P<0.01 vs. miR‑NC. (C) Relative expression of MCL1 in 57 AML bone marrow tissues and NBM was detected by RT‑qPCR. (D) Spearman's correlation analysis was performed to evaluate the correlation between MCL1 and miR‑193b‑3p expression. (E) Spearman's correlation analysis was performed to evaluate the correlation between MCL1 and SNHG14 expression. (F) Relative expression of miR‑193b‑3p in AML cell lines and human normal bone marrow CD34+ cells. **P<0.01 vs. CD34+ cells. The 2‑ΔΔCq method was used to analyse the relative mRNA expression level using GAPDH as an internal control. SNHG14, small nucleolar RNA host gene 14; miR, microRNA; NC, negative control; wt, wildtype; mut, mutated; NBM, normal marrow tissues; AML, acute myeloid leukaemia; MCL1, MCL1 apoptosis regulator BCL2 family member.

Article Snippet: Human normal bone marrow CD34+ cells and AML cell lines (MV‐4‐11, AML‐193, HL‐60, and KG‐1 cells) were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C.

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Expressing, Control, Negative Control

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: Figure 1. 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 ) retrie v ed 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 W ildt ype 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 e x cess Carboh y drates, ( M–M’ ) in e x cess lipids, ( N–N’ ) in e x cess proteins. Scale, 20 μm in all images. Individual dots represent ‘ n ’ of the sample. Significance w as e v aluated using tw o-w a y ANO V A with Tuk e y’s test w as perf ormed f or grouped analy ses. Error bar: standard de viation ( SD ) . Data are mean ± SD. * P < 0.033, ** P < 0.002 and *** P < 0.001. See also Supplementary Figure S1.

Article Snippet: Cell culture of human HSPC 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

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: Figure 5. Human blood progenitors exhibit leucine responsive proliferation similar to Drosophila . ( A–A’ ) EdU incorporation analysis of CD34+ CD10-Lin- m y eloid 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 reco v ery 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 ) Ly sotrack er 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 ) Quantitativ e analy sis of OPP incorporation rate at 6 and 16 h of leucine starv ation 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. Tw o-w a y ANOVA was performed for grouped analyses. One-way ANO V A 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 Supplementary Figure S5.

Article Snippet: Cell culture of human HSPC 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: Figure 6. 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 ph y sical 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 Supplementary Figure S6G ) . ( F ) EdU incorporation analysis upon leucine starvation and simultaneous treatment with B afilom y cinA1 ( B afA1 ) . ( 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 ) Quantitativ e analy sis of A go2 protein le v els upon leucine depriv ation and B afA1 treatment. ( I ) Schematic representing miRNA-9 sponge construct used for RNA-IP ( abo v e ) . Destabiliz ed 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 ANO V A 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: Cell culture of human HSPC 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