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: Scientific Reports

Article Title: Ocimum flavone Orientin as a countermeasure for thrombocytopenia

doi: 10.1038/s41598-018-23419-x

Figure Lengend Snippet: Effect of Orientin on human hematopoietic stem cells. ( a – c ) Orientin induces human CD34 + cells differentiation towards MEP lineage. CD34 + cells purified from healthy donor’s PBMCs via density gradient ficol followed by magnetic bead separation were treated with or without Orientin in a serum free expansion medium (SFEM) supplemented with 100 ng/ml cytokine cocktail (CC100) for 7 days. Cells were harvested for lineage differentiation and stained with CD34-PE-Cy7, CD38-Pacblue, CD10-FITC, CD135-PE, CD45RA-APC antibodies and subjected to FACS analysis. (d) Orientin partially restores CD34 + cells differentiation potential after irradiation. CD34 + cells purified from healthy donor’s PBMCs (1 × 10 5 /ml in triplicates) were treated with or without Orientin (5 µM) and exposed to 4 Gy (fractionated dose of 2 × 2 Gy). These cells were then co-cultured on human mesenchymal stromal cell layer in SFEM medium supplemented with 100 ng/ml cytokine cocktail for normal differentiation in 7 day time period. The MEP cells were gated in lin − CD34 + CD38 + CD10 − CD135 − CD45RA − block in each sample. ( e ) Colony Assay in CD34 + cells (1 × 10 3 /ml in triplicates) post 0 or 2 Gy X-ray exposure. CFU-GM and GEMM colonies were scored on day 14 by applying standard morphologic criteria.

Article Snippet: CD34 + cells were purified from PBMCs using Diamond CD34 isolation kit for human (#130-094-531, MiltenyiBiotec) as described by manufacturer.

Techniques: Purification, Staining, Irradiation, Cell Culture, Blocking Assay, Colony Assay

Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Stem cell transcriptome of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst MSC, hESC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Dendrogram showing hierarchical clustering of MSC, hESC and MSC-hiPSC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing MSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC (d) and MSC-hiPSC (e) is reported in the indicated tables [Fisher's exact test].

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Mesenchymal potential of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst different F-hiPSC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Principal Component Analysis (PCA) showing 3D visualization of Principal Component (PC) 1, PC2 and PC3 of differentially expressed genes for different F-hiPSC, MSC-hiPSC and hESC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing F-hiPSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC-hiPSC (d) and F-hiPSC (e) is reported in the indicated tables [Fisher's exact test]. f) Schematic of the differentiation protocol toward MSC-like cells. g) Representative images of adipogenic (A, scale bar is 50 µm), osteogenic (O, scale bar is 50 µm) and chondrogenic (C, scale bar is 400 µm) mesenchymal derivatives. h) Left panel: representative density plot showing CD45 + hematopoietic cells (P3) gated from total cells of the cobblestone area-forming cell assay; FSC-A, forward scatter area, a.u., arbitrary units. Right panel: representative histograms showing CD34 + hematopoietic progenitor subpopulation (purple) of CD45 + cells compared to unstained control (grey); the vertical axis represents event percentage count (Count%).

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test, Control

Journal: Journal of Nanobiotechnology

Article Title: Cerium oxide nanoparticles-carrying human umbilical cord mesenchymal stem cells counteract oxidative damage and facilitate tendon regeneration

doi: 10.1186/s12951-023-02125-5

Figure Lengend Snippet: Characterization of hUCMSCs. A Primary hUCMSCs were spindle-shaped under light microscopy. B Flow cytometry analysis showed that 98.5% of cells expressed CD29, 97.4% expressed CD44, 98.6% of cells expressed CD90, and 98.1% of cells expressed CD105. Meanwhile, only 0.48% and 0.28% expressed CD45 and CD34. C Lipid droplets were stained by oil red O after adipogenic differentiation induction of hUCMSCs. Mineralized nodules were stained by Alizarin Red after osteogenic differentiation induction of hUCMSCs. Acid mucopolysaccharide was stained by Alcian Blue after chondrogenic differentiation induction of hUCMSCs

Article Snippet: Briefly, 10 7 hUCMSCs were subjected to analysis for surface markers CD34 (E-AB-F1143D, Elabscience), CD44 (E-AB-F1100D, Elabscience), CD45 (E-AB-F1137D, Elabscience), CD29 (E-AB-F1049D, Elabscience), CD90 (E-AB-F1167D, Elabscience), and CD105 (E-AB-F1310D, Elabscience) by flow cytometry following the manufacturer’s protocol.

Techniques: Light Microscopy, Flow Cytometry, Staining

Journal: Molecular Therapy Nucleic Acids

Article Title: Ligand-modified rAAV6 vectors with nanoblades allow high level gene knock-in in HSPCs without compromising cell survival

doi: 10.1016/j.omtn.2025.102495

Figure Lengend Snippet: Figure 5. Coupling of rAAV6 with low amounts of mannose allowed high-level nanoblade-mediated knockin CD34+ cells without cell death induction (A) 8E4, 3E5, or 8E5 mannose sugars were incubated for 4 h at RT with 10E12 vg of rAAV6 vectors encoding for the donor template. 10E9 vg rAAV6 vector per condition were analyzed in dot blot using the ADK1 antibody for rAAV6 that recognizes the intact assembled capsid or conca- navalin A, which detected mannose bound to the capsid. (B) 5E8 vg of rAAV6 conjugated with indicated quantities of mannose were separated on a gel, followed by silver staining to reveal the intact capsids. (C and D) CD34+

Article Snippet: Purity of the selected CD34+ cell fraction was evaluated by fluorescence-activated cell sorting (FACS) analysis (FACSCanto, BD) with APC-conjugated anti-CD34 antibody (Miltenyi Biotech).

Techniques: Knock-In, Incubation, Plasmid Preparation, Dot Blot, Silver Staining