Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: The effect of statins on viability and growth of ( a ) stem ADMSC and non-cancerous HEK 293 cells and ( b ) cancer MiaPaCa-2 cells. ( a ) ADMSC—human adipose-derived mesenchymal stem cells, HEK 293—human embryonic kidney cells, exposure to statins—24 h, concentrations 0—100 µM, control—methanol, ( b ) previously published data , MiaPaCa-2—pancreatic cancer cells, exposure to statins—24 h, concentrations 0—40 µM, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of the effect of statins on the growth and viability of pancreatic cancer MiaPaCa-2 cells, non-cancerous HEK 293 cells, and ADMSC stem cells. Concentration of statins—20 µM, Time—exposure to statins—24, 48, and 72 h, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on size and compactness of spheroids. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr—methanol treated spheroids, P—pravastatin, R—rosuvastatin, L—lovastatin, F—fluvastatin, A—atorvastatin, Pi—pitavastatin, C—cerivastatin, S—simvastatin. Statins were added once, after spheroid formation, 10 weeks ( a ) or 3.5 weeks ( b ) after inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on the spheroid formation. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr methanol treated spheroids, P —pravastatin, R —rosuvastatin, L —lovastatin, F —fluvastatin, A —atorvastatin, Pi —pitavastatin, C —cerivastatin, S —simvastatin. Statins were added once, 24 h after cell inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of expression changes between statin treated and control MiaPaCa-2 and ADMSC cells. Displayed are only the genes that are differentially expressed upon at least one statin treatment in at least one cell type, requiring |log 2 FC|> 1 and FDR < 0.05. Statins were administered at a concentration of 12 µM for 24 h. ( FC fold change, FDR false discovery rate, horizontal and vertical axes—changes in ADMSC and MiaPaCa-2 cells, respectively, upon respective treatment). The red dashed lines indicate two-fold change increase or decrease in the gene expression. The genes with at least two-fold up-regulation (resp. down-regulation) in ADMSC stem cells are displayed to the right (resp. left) of the dashed lines. Similarly, genes with at least two-fold up-regulation (resp. down-regulation) in cancer cells are displayed above (resp. below) of the dashed lines. For details about differentially regulated transcripts see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579 .

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Expressing, Control, Concentration Assay, Gene Expression

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Cellular pathways most significantly affected by statins in cancer and stem cells. The gene set enrichment analysis (GSEA) revealed the KEGG pathways most affected by statin treatment in ADMSC and MiaPaCa-2 cells. Displayed is the union of the top five most enriched pathways among the comparisons. (Statin concentration—12 µM, treatment time—24 h, p-value—GSEA p-value, gene ratio—fraction of KEGG pathway genes among differentially expressed genes). For details about differentially regulated transcripts, see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control

Journal: Science Advances

Article Title: KRAS G12V /HLA-A*02:01–targeted chimeric antigen receptor T cells exhibit potent preclinical activity against solid tumors

doi: 10.1126/sciadv.aea2511

Figure Lengend Snippet: ( A ) Specificity screening of 11 positive clones against KRAS(WT)-HLA-A*02:01 and KRAS(G12V)-HLA-A*02:01 tetramers using enzyme-linked immunosorbent assay (ELISA; n = 3 independent experiments). ( B ) Antibody titration curves of selected clones, monoclonal antibody (mAb) A4 and B9, were measured by ELISA ( n = 3 independent replicates). OD 450 , optical density at 450 nm. ( C ) Schematic of CAR constructs showing CD19-, A4-, and B9-specific architectures. ( D ) Transduction efficiency was assessed on the basis of CD34 expression using flow cytometry ( n = 3 biological replicates). ( E ) Representative flow cytometry histograms showing CAR-positive populations after magnetic enrichment. ( F ) Frequency quantification of the indicated cell subsets by flow cytometry ( n = 3 biological replicates; ≥20,000 events analyzed per replicate). Data represent the means ± SD. Significance by two-way analysis of variance (ANOVA) with Tukey’s post hoc test: *** P < 0.001. TTE, terminally differentiated effector T cells; TEM, effector memory T cells; TCM, central memory T cells.

Article Snippet: CAR T cells were positively selected with the CD34 MicroBead Kit (Miltenyi Biotec, 130-100-453).

Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay, Titration, Construct, Transduction, Expressing, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Simultaneous Targeting of DNA Polymerase Theta and PARP1 or RAD52 Triggers Dual Synthetic Lethality in Homologous Recombination–Deficient Leukemia Cells

doi: 10.1158/1541-7786.MCR-22-1035

Figure Lengend Snippet: Targeting Polθ + PARP and Polθ + RAD52 induced dual synthetic lethality against HR-deficient leukemia cells. A, Western blot analysis showing the expression of indicated proteins in the nuclear fractions of Nalm6(RAD54+/+) and Nalm6(RAD54−/−) isogenic cell lines. B, HR activity in Nalm6(RAD54+/+) and Nalm6(RAD54−/−) cells: results represent mean % SD of GFP+ cells in RFP+ cells. Nalm6(RAD54+/+) and Nalm6(RAD54−/−) cells were treated for 72 hours (C) and 12 hours (D) with 25 mmol/L Polθi ART558, 2.5 nmol/L PARPi talazoparib, 10 mmol/L RAD52i 6-hydroxy-DL-dopa and the indicated combinations. Results show mean number SD of colony numbers (C) and mean % of tail DNA SD (D). E, Western blot analysis showing the expression of indicated proteins in the nuclear and cytosolic fractions of HEL cells with tet-inducible IDH1wt or IDH1(R132H) mutant. F, HR activity in HEL cells expressing IDH1wt or IDH1(R132H) mutant: results represent mean % SD of GFP+ cells in RFP+ cells. HEL cells expressing IDH1wt or IDH1(R132H) mutant were treated for 72 hours (G) and 12 hours (H) with 25 mmol/L Polθi ART558, 1 mmol/L PARPi olaparib, 2.5 mmol/L RAD52i 6-hydroxy-DL-dopa and the indicated combinations. Results show mean % SD of colony numbers when compared with untreated cells (G) and mean % of tail DNA SD (H). I, HR activity in Lin-CD34+ cells from HR-proficient and HR-deficient AMLs (n = 3 of each). Results represent mean % SD of GFP+ cells in RFP+ cells. J, Lin-CD34+ cells from HR-proficient and HR-deficient AMLs (n = 3 of each) were treated with 25 mmol/L Polθi ART558, 1.25 mmol/L PARPi olaparib, 2.5 mmol/L RAD52i 6-hydroxy-DL–dopa and the indicated combinations. Results represent mean % SD of colonies in comparison with untreated cells. K, Survival curves of the AML mice treated with vehicle (C), Polθi RP6685, PARPi olaparib, and the combination. (B–D and F–J) Statistical significance when compared with: # another group, + control, and * corresponding individual treatments. K, ***, P < 0.001 in comparison to other groups.

Article Snippet: Lin − CD34 + cells were obtained from mononuclear fractions by magnetic sorting using the EasySep negative selection human progenitor cell enrichment cocktail followed by human CD34 positive selection cocktail (Stemcell Technologies) as described before ( 3 ).

Techniques: Western Blot, Expressing, Activity Assay, Mutagenesis, Comparison, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients.

doi: 10.1038/sj.mt.6300353

Figure Lengend Snippet: Figure 1 Human CD45+ cell engraftment and B-cell development in the bone marrow (BM) of IL2RβAb/NOD-SCID mice transplanted with trans- duced Artemis- or RAG1-deficient CD34+ cells. Artemis- or RAG1-deficient CD34+ cells were cultured in the presence of cytokines and transduced (T) or not transduced (NT) with Artemis- or RAG1-expressing lentiviral vectors. Control CD34+ cells were cultured under the same conditions. NOD-SCID mice were irradiated and pre-treated with IL2RβΑb/NOD-SCID prior transplantation with human cells. Eight weeks later, mice were killed and fluorescence- activated cell sorting analyses were performed on the BM cells to evaluate: (a) the level of human cell engraftment (percentage of CD45+ cells) and (b, c) B-cell development status (percentage of CD19+ cells) in the human CD45+ population and percentage of CD19+IgM+ cells in the CD45+ subset. Each point represents an individual with more than 5% of human CD45+ cells in its BM. Red, Green, Black, and open triangles indicates mice engrafted with control CD34+ cells (ctrl, n = 4); Mice were engrafted with non-transduced (NTArtemis, n = 4) or transduced (TArtemis, n = 5) Artemis-deficient CD34+ cells from patients A1(blue squares), A2 (green and open squares), A3 (red squares) and A4 (black squares). Mice were engrafted with non-transduced (NTRAG1, n = 4) or transduced (TRAG1, n = 8) RAG1-deficient CD34+ cells from patients R1 (black circles), R2 (open circles), R3 (yellow, red, and green cir- cles), R5 (pink, tan, and blue circles). Horizontal line indicates mean values. *The difference between the two groups was statistically significant (P < 0.05), according to the Mann–Whitney U-test. (d) B-cell reconstitution was analyzed in BM cells from NOD-SCID mice transplanted with transduced CD34+ cells from patients A4 or R1 or with control cells. The dot-plot analysis was performed on the gated human CD45+ population. IgM, immunoglobulin M.

Article Snippet: CD34+ cells were positively selected using the human Miltenyi indirect CD34 Microbead Kit (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Cell Culture, Expressing, Control, Irradiation, Transplantation Assay, Fluorescence, FACS, MANN-WHITNEY

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients.

doi: 10.1038/sj.mt.6300353

Figure Lengend Snippet: Figure 3 Restoration of B-cell function after lentiviral transduction of CD34+ cells isolated from RAG1- or Artemis-deficient bone mar- row (BM) samples. (a) Human IgMs were quantified in the serum of IL2RβΑb/NOD-SCID mice successfully engrafted with T or NT Artemis- or RAG1-deficient CD34+ cells. Each circle represents an individual mouse—indicates mean values. (b) Using polymerase chain reaction and GeneScan analysis, complete V(D)J rearrangements were evaluated in the BM and spleen of control CD34+ cells and transduced or non- transduced Artemis-deficient CD34+ cells from patient A4 (NTArtemis and TArtemis, respectively. The Gaussian curves confirmed the presence of polyclonal V(D)J rearrangements. The use of three differently labeled primers allowed identification of the JH segments used and gave better resolution of the polyclonal rearrangements, as shown. Size markers are in red. IgM, immunoglobulin M; ND, not determined.

Article Snippet: CD34+ cells were positively selected using the human Miltenyi indirect CD34 Microbead Kit (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Cell Function Assay, Transduction, Isolation, Polymerase Chain Reaction, Control, Labeling

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients.

doi: 10.1038/sj.mt.6300353

Figure Lengend Snippet: Figure 2 Human CD45+ cell engraftment and B-cell development in the spleen of non-obese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted with transduced Artemis-deficient CD34+ cells. Fluorescence-activated cell sorting analysis was performed on splenocytes isolated from IL2RβAb/NOD-SCID mice transplanted with TArtemis- or NTArtemis-deficient CD34+ cells or with control cells. Each point represents (a) the percentage of CD45+ cells, (b) the percentage of CD45+CD19+ cells and (c) the percentage of CD19+IgM+ cells in the CD45+ subset. Black, red, green, and open triangles indicates mice engrafted with control CD34+ cells (ctrl, n = 4); Mice were engrafted with non-transduced (NTArtemis, n = 4) or transduced (TArtemis, n = 5) Artemis-deficient CD34+ cells from patients A1 (blue squares), A2 (green and open squares), A3 (red squares), and A4 (black squares). *The dif- ference between the two groups was statistically significant (P < 0.05), according to the Mann–Whitney U-test. Horizontal square indicates mean values. (d) Analysis of the presence of human B cells in the spleen of a NOD-SCID mice transplanted with T or NT-deficient CD34+ cells from patient A4. The dot-plot analysis was performed on the gated human CD45+ population.

Article Snippet: CD34+ cells were positively selected using the human Miltenyi indirect CD34 Microbead Kit (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Fluorescence, FACS, Isolation, Control, MANN-WHITNEY