Journal: Frontiers in Immunology

Article Title: Pro-inflammatory-Related Loss of CXCL12 Niche Promotes Acute Lymphoblastic Leukemic Progression at the Expense of Normal Lymphopoiesis

doi: 10.3389/fimmu.2016.00666

Figure Lengend Snippet: Biological characterization of mesenchymal stromal cells (MSC) derived from bone marrow of ALL patients . MSC were evaluated according to morphology (A) , colony-forming unit-fibroblast capacity (B) , proliferation rates by carboxifluorescein dilution assay (C) , multiple differentiation potential ( N NBM = 5, N ALL = 7) (D) , minimal criteria immunophenotype ( N NBM = 5, N ALL = 7) (E) , and differentiation genes expression ( N NBM = 4, N ALL = 5) (F) . NBM, normal bone marrow; ALL, acute lymphoblastic leukemia.

Article Snippet: CD34 + cells from normal bone marrow (NBM) and ALL were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Dilution Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Pro-inflammatory-Related Loss of CXCL12 Niche Promotes Acute Lymphoblastic Leukemic Progression at the Expense of Normal Lymphopoiesis

doi: 10.3389/fimmu.2016.00666

Figure Lengend Snippet: Acute lymphoblastic leukemia (ALL)-mesenchymal stromal cells (MSC) contribute to leukemic cell maintenance by creating a pro-inflammatory microenvironment . Hematopoietic stem and progenitor cells (HSPC) from ALL bone marrow were cocultured on normal bone marrow (NBM)-MSC or ALL-MSC monolayers in lymphoid conditions during 4 weeks before flow cytometry analysis of the newly produced cells, and the B cell yield per input were recorded ( N NBM = 5, N ALL = 7) (A) . Cytokines, chemokines, and growth factors production by NBM and ALL-MSC were evaluated after collection of 24-h supernatants. Data are normalized to NBM-MSC secretion (B) ( N NBM = 5, N ALL = 13). Phosphorylated p65 (NF-κB) was evaluated by immunofluorescence microscopy ( N NBM = 3, N ALL = 4) [ (C) , left panel] and flow cytometry [ (C) , left panel] of MSC ( N NBM = 3, N ALL = 5). Normalized nuclear fluorescence intensity and mean fluorescence intensity (MFI) from flow cytometry are shown.

Article Snippet: CD34 + cells from normal bone marrow (NBM) and ALL were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Produced, Immunofluorescence, Microscopy, Fluorescence

Journal: Frontiers in Immunology

Article Title: Pro-inflammatory-Related Loss of CXCL12 Niche Promotes Acute Lymphoblastic Leukemic Progression at the Expense of Normal Lymphopoiesis

doi: 10.3389/fimmu.2016.00666

Figure Lengend Snippet: Remodeling of the lymphoid normal niche is associated with leukemic colonization concomitant to displacement of normal progenitors . Stromal spheroids were cocultured with acute lymphoblastic leukemia (ALL) primary cells (A) . The colonization was confirmed by immunofluorescence microscopy after staining CD34 + cells with fluorescent dyes red and blue (B) . Viability of leukemic cells harvested from 2D and tridimensional (3D) cocultures was assessed by flow cytometry after 7 days (C) . CXCL12 was evaluated by flow cytometry in 3D MSC spheroids after enzymatic digestion compared with 2D monolayer cultures (D) . CD34 + cells from mobilized peripheral blood and CD34 + ALL cells were cocultured in a competence ratio 1:1 for 24 h in normal bone marrow (NBM) and ALL mesenchymal stromal cells (MSC) spheroids (E) or during 120 h in NBM-MSC spheroids (F) . Spheroids were washed, and CXCL12 expression (green) was evaluated ( N NBM-CD34 + = 2 , N ALL-CD34 + = 2 , N NBM-MSC = 3, N ALL-MSC = 4).

Article Snippet: CD34 + cells from normal bone marrow (NBM) and ALL were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Microscopy, Staining, Flow Cytometry, Expressing

Journal: Frontiers in Oncology

Article Title: The miR-372 -ZBTB7A Oncogenic Axis Suppresses TRAIL-R2 Associated Drug Sensitivity in Oral Carcinoma

doi: 10.3389/fonc.2020.00047

Figure Lengend Snippet: ZBTB7A expression is associated with apoptosis and drug sensitivity in OSCC cells. (A–G,I–L) SAS cells. (H) FaDu cells. (A) Lt, Increased miR-372 expression in CDDP resistance (CDDP-R) and taxol resistance (taxol-R) SAS cell subclones relative to the parental cells. Rt, ZBTB7A expression is slightly downregulated in these cell subclones. (B,C) Apoptosis assay. Cells with transient ZBTB7A knockdown (in B , Lt), as well as the stable knockdown cell subclone (in C ), were treated with CDDP or taxol to induce apoptosis. (B) Rt, quantification of the presence of apoptotic cells. Lt, ZBTB7A knockdown decreases the apoptosis induced by the above drugs. (D) Quantification of the apoptotic cells. CDDP-induced apoptosis is enhanced by 3MA treatment, but not by Ferrostatin-1 treatment. Taxol-induced apoptosis is slightly enhanced by 3MA treatment, but not by Ferrostatin-1 treatment. (E) Western blot analysis. Differential ZBTB7A expression in sh-ZBTB7A (7332) knockdown, ZBTB7A exogenous expression and both knockdown and expression subclones. (F–H) Cell viability assays. (F) Upper, CDDP. Lower, taxol. These shows an association between ZBTB7A expression level and drug sensitivity. (G,H) The results show that ZBTB7A expression sensitizes SAS cells to AG1478 treatment. miR-372 expression and ZBTB7A knockdown are associated with CDDP resistance in both SAS and FaDu cells. (I,J) 3D culture of SAS cells. (I) Representative colonies undergoing 3D culture. CDDP disrupts the colonies, while knockdown of ZBTB7A partly restores the integrity of colonies. Bars, 1 mm. (J) Quantification of cell viability during 3D culture. Lt, ZBTB7A knockdown. Rt, ZBTB7A exogenous expression. The results show that there is an association between ZBTB7A expression and CDDP sensitivity during 3D culture. (K) Mitochondrial membrane potential analysis. Lt, flow cytometry diagrams. The cells were treated with 15 or 30 μM CDDP for 48 h then stained with JC-1. The shift in fluorescence from red to green indicates the collapse of mitochondrial membrane potential. Rt, Quantitative analysis of red/green ratio. CDDP reduces the ratio in a dose-dependent manner, while ZBTB7A knockdown reverses this change. (L) Subcutaneous tumorigenicity. ZBTB7A expression decreases xenografic growth, while the CDDP regimen further increases the inhibitory efficacy of ZBTB7A. ns , not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A Human Apoptosis Antibody Array Kit (ARY009; R&D Systems, Minneapolis, MN) was used to verify the presence of various proteins known to be crucial to apoptosis.

Techniques: Expressing, Apoptosis Assay, Knockdown, Western Blot, Membrane, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Oncology

Article Title: The miR-372 -ZBTB7A Oncogenic Axis Suppresses TRAIL-R2 Associated Drug Sensitivity in Oral Carcinoma

doi: 10.3389/fonc.2020.00047

Figure Lengend Snippet: ZBTB7A expression is associated with the expression of death receptors and the phosphorylated isoforms of p53 in OSCC cells. (A,B,D) SAS cells. (A) Apoptosis array. A panel of 35 antibodies including various forms of phosphorylated p53 were measured in duplicate using this array. The ZBTB7A knockdown cell subclone and the control cells were treated with CDDP for 24 h, and then the array was used to identify changes in various apoptosis factors. Lt, a diagram of the array. White rectangles indicate duplicates exhibiting significant changes in signal following ZBTB7A knockdown. Rt, a heat-map demonstrating the changes in protein profile following ZBTB7A knockdown. The 3rd lane represents the average value of the duplicate signals in the upper two lanes. Vertical lines define the 20 proteins that are downregulated >30% (Lt region) or that are upregulated >12% (Rt region). (B) RNA sequencing. A heat-map showing the changes in transcript levels in the ZBTB7A subclone. The genes selected for analysis are TRAIL-R1, TRAIL-R2, Fas and the various phosphorylated isoforms of p53 and many of these were downregulated following knockdown of ZBTB7A; thus these are to be considered as ZBTB7A targets during apoptosis modulation. (C,D) Western blot analysis. (C) Lt, SAS cells, Rt, FaDu cells. Expression of TRAIL-R1, TRAIL-R2, Fas, and p53 phosphorylated at serine 15 is correlated with ZBTB7A expression (in C ), and is inversely correlated with ZBTB7A knockdown (in C ) and miR-372 expression (in D ) in OSCC cells. (E) qRT-PCR analysis. This shows the concordant changes in TRAIL-R1 and TRAIL-R2 mRNA expression in OSCC cells following ZBTB7A knockdown/expression. (F,G) Promoter activity assay. This shows the consistent increase and decrease in TRAIL-R2 activity that follows expression and transient knockdown of ZBTB7A in OSCC cells, respectively. ns , not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A Human Apoptosis Antibody Array Kit (ARY009; R&D Systems, Minneapolis, MN) was used to verify the presence of various proteins known to be crucial to apoptosis.

Techniques: Expressing, Knockdown, Control, RNA Sequencing, Western Blot, Quantitative RT-PCR, Activity Assay

Journal: Frontiers in Oncology

Article Title: The miR-372 -ZBTB7A Oncogenic Axis Suppresses TRAIL-R2 Associated Drug Sensitivity in Oral Carcinoma

doi: 10.3389/fonc.2020.00047

Figure Lengend Snippet: ZBTB7A sensitizes SAS cells to CDDP by trans-activation TRAIL-R2. (A) Lt Upper, schematic diagram depicting the ChIP strategy. R2-1 and R2-2 are predicted to be ZBTB7A binding sites in the TRAIL-R2 promoter. ( A , Lt Lower; B , Lt) Representative gel electrophoresis images of the PCR analysis; these reveal the amplicons of R2-1 and R2-2 sequences using the various immunoprecipitates, namely control, ZBTB7A knockdown and ZBTB7A exogenous expression cells. ( A,B , Rt), quantification. The analysis indicates that ZBTB7A expression increases, and ZBTB7A knockdown decreases the binding of the ZBTB7A to TRAIL-R2-1 and TRAIL-R2-2 sites, respectively. Anti-Z: anti-ZBTB7A antibody. (C,D) Long-form and short-form TRAIL-R2 expression, respectively. Lt, Western blot analysis confirming the expression of TRAIL-R2 in the SAS ZBTB7A knockdown cell subclone by means of TRAIL-R2 retroviral infection. Rt, CDDP sensitivity as related to expression of ZBTB7A and TRAIL-R2. (E) Apoptosis assay. Lt, flow cytometry diagram. Rt, quantification. The CDDP-induced apoptosis is decreased by ZBTB7A knockdown and this is reversed by TRAIL-R2 expression. (F) Lt, Mitochondrial membrane potential analysis. Rt, Quantification of the red/green ratio. Following 15 or 30 μM CDDP for 48 h, the increase in the red/green ratio as a result of ZBTB7A knockdown is reversed by TRAIL-R2 expression. (G) Western blot analysis of paired OSCC tissue samples. TRAIL-R2 expression is clearly downregulated in the OSCC tissue samples. N, NCMT; T, OSCC. (H) Kaplan-Meier analysis of the overall survival in relation to ZBTB7A copy number and TRAIL-R2 copy number. ns , not significant; * p < 0.05; ** p < 0.01.

Article Snippet: A Human Apoptosis Antibody Array Kit (ARY009; R&D Systems, Minneapolis, MN) was used to verify the presence of various proteins known to be crucial to apoptosis.

Techniques: Activation Assay, Binding Assay, Nucleic Acid Electrophoresis, Control, Knockdown, Expressing, Western Blot, Retroviral, Infection, Apoptosis Assay, Flow Cytometry, Membrane

Journal: Frontiers in Oncology

Article Title: The miR-372 -ZBTB7A Oncogenic Axis Suppresses TRAIL-R2 Associated Drug Sensitivity in Oral Carcinoma

doi: 10.3389/fonc.2020.00047

Figure Lengend Snippet: miR-372 associated oncogenicity and CDDP resistance in SAS cells. (A) 5′sgRNA and 3′sgRNA are designed to delete hsa- miR-372 -3p. (B) PCR analysis is used to screen the deletion cell subclones. The dotted line indicates the ~300-bp position on the gel electrophoresis image. Cell subclones exhibiting a band below the dotted line and the absence other bands (marked by triangles) are suspected to have a homozygous deletion. (C) qRT-PCR analysis. This reveals almost complete absence of miR-372 expression in the sublcones selected in (B) . (D) Western blot analysis revealing the upregulation of ZBTB7A, TRAIL-R1, and TRAIL-R2 expression in nearly all subclones except for subclone #34. (E,F) Invasion assay and anchorage-independent colony formation assay, respectively. The assays show the general decrease of these properties in the deleted subclones that are tested. (G) Flow cytometry analysis to detect apoptosis. The CDDP induced apoptosis is higher in deleted subclones relative to the control cells. (H) Cell viability assay. The miR-372 deleted cell subclones exhibit higher sensitivity to CDDP. ns , not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: A Human Apoptosis Antibody Array Kit (ARY009; R&D Systems, Minneapolis, MN) was used to verify the presence of various proteins known to be crucial to apoptosis.

Techniques: Nucleic Acid Electrophoresis, Quantitative RT-PCR, Expressing, Western Blot, Invasion Assay, Colony Assay, Flow Cytometry, Control, Viability Assay

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 1. The expressions of PD-L1 in human preeclampsia (PE) placentas (n = 30) and normal pregnant women (n = 30). (A,B) PD-1 levels were significantly reduced in PE placentas. (C,D) PD-L1 levels were overtly reduced in pre-eclamptic mothers. (E,F) GM-CSF levels were prominently decreased in the PE set. Relative JAK2 (G) and STAT5 (H) protein levels were markedly lessened, while those of p-JAK2 (I) and p-STAT5 (J) were significantly enriched in pre-eclamptic placentas. (K) Western blotting results of related molecules were shown. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 2. The transfection efficiency in cell lines and the changes in expressions of relevant factors after lentivirus treatment (n = 3, for each group). (A,B) The valid transfection efficiency in the two cell lines was confirmed by the presence of green fluorescence. Scale bars, 100 μm. The mRNA (C,L) and protein (D,M) levels of PD-L1 were significantly reduced after the knockdown of PD-L1 in two cell systems. The mRNA (E,N) and protein (F,O) levels of GM-CSF were notably lower after transfection with siR-PD-L1 in the two trophoblasts. JAK2 and STAT5 protein expressions were induced, while their phosphorylation expressions were inhibited in HTR- 8/SVneo (G–J) and JEG-3 (P–S) cells with the introduction of LV-PD-L1. (K,T) Western blot of PD-L1, GM- CSF, and JAK2/STAT5 molecules was carried out in the two cell lines. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Transfection, Fluorescence, Knockdown, Phospho-proteomics, Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 4. Influences of aberrant GM-CSF on PD-L1 and the JAK2/STAT5 pathway (n = 3, for each group). The protein levels (A,H) of PD-L1 were evidently elevated after adding GM-CSF in trophoblasts. GM-CSF protein levels (B,I) were significantly higher by the accumulation of GM-CSF in the two trophoblasts. The levels of JAK2 and STAT5 were overtly lower, while their phosphorylation levels were enhanced exposure to the GM- CSF neutralizing antibody in HTR-8/SVneo (C–F) and JEG-3 (J–M) cells. (G,N) Western blot bands were displayed in the two cell lines. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Phospho-proteomics, Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 5. The biological role of GM-CSF in the cell lines (n = 3, for each group). (A,B)Over-regulation of GM- CSF elevated the migratory property of cells. (C,D) GM-CSF stimulation increased cell invasion. Scale bar, 500 μm. (E,F) GM-CSF up-regulation suppressed cell apoptosis. (G–I) Increased GM-CSF promoted the evolvement of cell cycle from the G1 to the S stage in both cells. (J) (K) Cells supplemented with GM-CSF exhibited higher proliferative ability in trophoblasts. ns, no significance. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques:

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 6. The rescue assay and the impact of the JAK2 inhibitor on regulating GM-CSF (n = 3, for each group). (A,B) The rescue assay of co-transfection of PD-L1 and GM-CSF in two kinds of trophoblasts. The protein levels (C,H) of GM-CSF were elevated with the JAK2 inhibitor. JAK2 (D,I) and STAT5 (E,J) protein levels were not influenced by the JAK2 inhibitor. The levels of p-JAK2 (F,K) and p-STAT5 (G,L) were profoundly inhibited by the addition of JAK2 inhibitor. (M) The bands of western blot in rescue experiments. (N) Western blotting was shown in cells with the JAK2 inhibitor. ns, no significance. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Rescue Assay, Cotransfection, Western Blot

Journal: Scientific reports

Article Title: Decreased PD-L1 contributes to preeclampsia by suppressing GM-CSF via the JAK2/STAT5 signal pathway.

doi: 10.1038/s41598-025-87349-1

Figure Lengend Snippet: Fig. 7. The blood pressure, urine protein, and the expressions of related indicators in different groups of pregnant rats (n = 5, for each group). (A,B) LV.PD-L1 reduced the blood pressure of PE-like animals on GD17 and GD19. (C) LV.PD-L1 significantly decreased the urinary protein content on GD19. Fetal weight (D), fetal length (E), and placental weight (F) in different groups. (G) Western blot in rat placental tissues. (H,I) The expressions of PD-1 were evidently reduced after L-NAME treatment. (J,K) The levels of PD-L1 were notably decreased in placentas from PE-like pregnant rats. (L,M) The expressions of GM-CSF were elevated following adding LV.PD-L1. (N–Q) The relative band intensity of the JAK2/STAT5 pathway was compared using quantification. DAPI staining and the green fluorescence representing viral infection were displayed in the L-NAME + PD-L1 NC (R) and L-NAME + LV.PD-L1 (S) sets. Scale bar, 50 μm. Data are analyzed by χ2-test between the two groups.

Article Snippet: In order to test the influence of GM-CSF on the functions of trophoblasts, cell lines were added with 100 ng/ ml recombinant human GM-CSF protein (Peprotech, Rocky Hill, NJ), 10 μg/ml neutralizing GM-CSF antibody, or 10 μg/ml GM-CSF isotype control mAb (Proteintech Group, Rosemont, IL, United States) for 48 h. Similar to another experimental proposal33, recombinant GM-CSF protein was used to treat one set of cell lines, while another set of cell lines did not receive this treatment.

Techniques: Western Blot, Staining, Fluorescence, Infection

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet:

Article Snippet: Human GM-CSF ELISA Kit , R&D Systems , #DY215-05.

Techniques: Virus, Microarray, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Plasmid Preparation, Expressing, Software

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: List of primers and corresponding annealing temperatures for RT 2 -PCR.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Sequencing

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 confers TMZ-resistance on GBM cells. ( A ) Confirmation of PDCD10 knockdown in lentiviral transduced U87 and T98g cells by RT 2 -PCR ( a ), western blot ( b ), and semi-quantitation of the blots ( c ). ev and sh: empty vector- and PDCD10 shRNA-transduced cells, respectively. IOD: integrated optical density. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev. ( B ) Knockdown of PDCD10 in GBM cells leads to a resistance to TMZ-induced cell death. U87 and T98g cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h. Thereafter, TMZ was washed-out. Remaining viable cells were cultured in the TMZ-free medium for 3 d, which is defined as the post-treatment phase. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). MTT assay was performed to determine the viability of cells at 72 h after TMZ treatment (treatment phase) and 3 d after washing-out of TMZ (post-treatment phase). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev; ##, p < 0.01; ###, p < 0.0001, compared with evC in the same phase; +++, p < 0.001, compared with corresponding group in treatment phase.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Western Blot, Quantitation Assay, Plasmid Preparation, shRNA, Cell Culture, Control, MTT Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 enhances cell viability after rechallenge with TMZ in regrown cells (RG) generated from the established acquired TMZ-resistant model. ( A ) Confirmation of PDCD10 knockdown in shU87-RG and shT98g-RG cells by RT 2 -PCR ( a ) and by FACS of respective transduced cells that expressed red-fluorescence protein (RFP) and green-fluorescence protein (GFP) ( b ). *, p < 0.05, compared with ev. ( B ) MTT assay in RG cells in treatment phase and post-treatment phase. MTT assay was performed with ev/shU87 and ev/shT98g cells that received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h (treatment phase) and 2 d and 4 d after washing-out TMZ (post-treatment phase, without reseeding). Control cells (C) received vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; ***, p < 0.001, compared with evRG C (72 h); ###, p < 0.001, compared with evRG-TMZ (72 h). ( C ) MTT assay in RG cells in a second post-treatment model with reseeding. ev/shU87-RG and ev/shT98g-RG cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h. Thereafter, TMZ–containing media and dead cells were washed-out, and the viable cells were harvested and reseeded at the same density, followed by 2, 4, and 6 d of culture in drug-free medium. A significantly more rapid regrowth was observed in both TMZ-treated shU87-RG and shT98g-RG cells, compared with the corresponding evRG cells after reseeding and culturing in drug-free media. *, p < 0.05; ***, p < 0.001, compared with corresponding evRG.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Generated, Fluorescence, MTT Assay, Control

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Overexpression of PDCD10 sensitizes GBM cells to TMZ treatment. ( A ) Confirmation of overexpression of PDCD10 in lentiviral transduced U87 and T98g cells by RT 2 -PCR ( a ) and western blot ( b ) and semi-quantitation of the blots ( c ). Western blotting with anti-V5 antibody distinguishes between the expression of transgenic C-terminal V5-tagged PDCD10 protein and endogenous protein. ev and ox: empty vector-transduced and PDCD10-overexpressing cells, respectively. IOD: integrated optical density. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev. ( B ) Overexpression of PDCD10 significantly reduces cell viability in a concentration-dependent manner after 72 h of TMZ treatment in both oxU87 ( a ) and oxT98g ( b ) cells. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev groups. ( C ) Overexpression of PDCD10 sensitizes GBM cells to TMZ treatment 72 h after TMZ treatment (treatment phase) and 3 d after washing-out TMZ (post-treatment phase). ev/oxU87 and ev/oxT98g cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ for 72 h, respectively. Thereafter, TMZ-containing medium and dead cells were washed-out and the viable cells were further cultured in drug-free medium for 3 d followed by MTT assay. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev groups; +++, p < 0.001, compared with corresponding evC in the treatment phase; #, p < 0.05, ###, p < 0.001, compared with corresponding evC in the same phase.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Transgenic Assay, Plasmid Preparation, Concentration Assay, Control, Cell Culture, MTT Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: DNA replication in response to TMZ treatment is dependent on PDCD10 expression. DNA replication was detected by EdU incorporation followed by FACS at 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and -culturing in drug-washout media (post-treatment phase). ev/shT98g-RG and ev/oxT98g cells received 500 and 300 µM TMZ, respectively. ( A , C ) Histograms of EdU-positive (EdU+) and -negative (EdU−) cell populations in ev/shT98g-RG and ev/oxT98g cells, respectively. ( B , D ) Bar graphs of EdU+/− populations based on the corresponding histograms in ( A , C ). Knockdown of PDCD10 leads to an increase in DNA replication in both the treatment and post-treatment phases of T98g-RG cells, whereas overexpression of PDCD10 suppresses DNA replication in response to TMZ treatment. The data are representative of at least three independent experiments.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Expressing, Knockdown, Over Expression

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Alteration in cell cycle checkpoints in response to TMZ treatment is dependent on PDCD10 expression. Cell cycle assay was performed by FACS after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and -culturing in drug-washout media (post-treatment phase). ev/shT98g-RG and ev/oxT98g cells received 500 and 300 µM TMZ, respectively. ( A , C ) are representative of cell cycle histograms in ev/shT98g-RG and ev/oxT98g cells, respectively. DNA content-based cell cycle distributions were defined using FlowJo with the Dean–Jett–Fox algorithm and presented in histograms. Each cell cycle phase is shown in different colors: G0/G1- (blue), S- (yellow), and G2/M-phase (green). ( B , D ) Stacked bar graphs of the distribution of the cell population in the cell cycle based on the corresponding histograms in ( A , C ). Knockdown of PDCD10 leads to the escape of cells from G2/M arrest and increases the population in the S phase (DNA replication phase) in both the treatment and post-treatment phases, whereas overexpression does the opposite. The data are representative of at least three independent experiments.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Expressing, Cell Cycle Assay, Knockdown, Over Expression

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 in T98g-RG cells leads to deregulation of DNA damage response (DDR) genes. ev/shT98g-RG and ev/oxT98g cells received 300 µM of TMZ or vehicle (0.2% DMSO) treatment (no TMZ treatment). Cells were harvested for PCR detection of DDR genes after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and culturing in drug-free media (post-treatment phase). ( A ) Expression of MGMT in T98g-RG ( a ) cells and ev/oxT98g ( b ) cells in the no treatment, treatment (300 µM, 72 h), and post-treatment (3 d after washing out) phases. ( B ) Western blot ( a ) and semi-quantitation of the blots ( b ) of the MGMT protein expression in ev/shT98g-RG and ev/oxT98g cells. ( C ) Expression of DDR genes ( MSH2 , MSH6, and PMS2 ) in T98g-RG cells in the no treatment ( a ), treatment ( b ), and post-treatment phases ( c ), respectively. ( D ) Expression of DDR genes in ev/oxT98g cells in the no treatment ( a ), treatment ( b ), and post-treatment phases ( c ), respectively. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with corresponding ev.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Expressing, Western Blot, Quantitation Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: PDCD10 expression determines the colony formation capacity of GBM cells. ev/shT98g-RG ( A ) and ev/oxT98g ( B ) cells received 300 µM of TMZ or vehicle (0.2% DMSO) treatment. Cells were harvested for colony formation assay in a 12-well plate in triplicate after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and culturing in drug-free media (post-treatment phase). The number of colonies was quantified after staining with 0.5% crystal violet using the ImageJ software (version 1.54j). Representative images of colony formation in ev/shT98g-RG and ev/oxT98g are shown in ( Aa ) and ( Ba ), respectively. Quantitative analysis of the colony numbers is presented in ( Ab ) and ( Bb ) for ev/shT98g-RG and ev/oxT98g cells, respectively. ***, p < 0.001, compared with ev; ##, p < 0.01 and ###, p < 0.001, compared with corresponding C; +++, p < 0.001, compared with corresponding group in the treatment phase.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Expressing, Colony Assay, Staining, Software

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 enhances the self-renewal capacity of U87-RG cells and GSCs generated from the parental cell line U87-RG (U87-RG-GSCs), and increases the expression of stem cell markers in RG-GSCs. ( A ) Representative images of neurospheres derived from U87-RG cells and U87-RG-GSCs. Scale bar: 100 µm. ( B ) Quantitative analysis of neurospheres formation efficiency (SFE). ***, p < 0.001, compared with corresponding ev; ###, p < 0.001, compared with corresponding parental cells. ( C ) Knockdown of PDCD10 increases the mRNA expression of stemness genes in RG-GSCs. The expression of stem cell markers Nestin and KLF4 was detected by RT 2 -PCR in untreated U87-RG-GSCs, and in shU87-RG-GSCs treated with TMZ (150 µM) for 72 h. *, p < 0.05; ***, p < 0.001, compared with corresponding ev; #, p < 0.05; ###, p < 0.001, compared with evC; +, p < 0.05, compared with corresponding C. ( D ) Knockdown of PDCD10 enhances the viability of parental U87-RG cells and their GSC variants. U87-RG cells (left) and U87-RG-GSCs (right) received TMZ (150 M) or vehicle DMSO (0.1%) treatment for 72 h. Cell viability was detected after 72 h of treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev; #, p < 0.05; ##, p < 0.01, compared with evC; +, p < 0.05, ++, p < 0.01, compared with corresponding parental cells. ( E ) Knockdown of PDCD10 reduces the mRNA expression of MMR genes ( MSH2 , MSH6 , and PMS2 ) in U87-RG-GSCs. U87-RG-GSCs received treatment of 150 µM TMZ or vehicle (0.1% DMSO; no treatment). Cells were harvested for PCR detection of MMR genes after 72 h of treatment. Expression of MMR genes in non-treated ( Ea ) and TMZ-treated U87-RG-GSCs ( Eb ). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Generated, Expressing, Derivative Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Schematic summary of the role and mechanism of PDCD10 in acquired TMZ-resistance. Knockdown of PDCD10 (shPDCD10) in GBM cells significantly increased cell survival in response to TMZ treatment, and strongly promoted tumor cell regrowth in the post-treatment phase, which collectively accounted for acquired TMZ-resistance. Mechanism studies revealed that the loss of PDCD10 modulated the expression of DNA damage response genes (i.e., upregulating MGMT and downregulating MMR genes MSH2 , MSH6 , and PMS2 ), and altered the cell cycle process, as evidenced by the evasion of tumor cells from arrest at the G2/M phase, and the increase in tumor cells in the proliferating S phase. In addition, shPDCD10-GBM cells exhibited higher cell plasticity, as demonstrated by an increased capacity for colony formation and transformation of shPDCD10-GBM cells into GSC-like cells that expressed higher levels of the stem cell markers Nestin and KLF4. In support of these findings, overexpression of PDCD10 (oxPDCD10) induced contrary changes in the molecular and cell behaviors observed in shPDCD10-GBM cells, increasing the sensitivity of oxPDCD10-GBM cells to TMZ treatment and suppressing tumor cell regrowth after TMZ treatment. Our results indicate that PDCD10 plays a pivotal role in acquired TMZ-resistance and thus represents a promising target for perturbing TMZ-resistance and tumor recurrence.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Expressing, Transformation Assay, Over Expression

Journal: bioRxiv

Article Title: Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

doi: 10.1101/2025.04.14.648845

Figure Lengend Snippet: A. Schematic of genomic editing strategy at the HBA1 locus for targeted integration of CD4 expression cassettes delivered by AAV6. B. Percentage of alleles with targeted integration for each CD4-expression cassette. Each AAV6 was used at 2500 vector genomes/cell. n=6 independent HSPC donors for Mock, CD4-GPA, CD4-GPA-tEPOR, n=4 for CD4 and CD4-tEPOR. Bars represent median +/− 95% confidence interval. C. CFU assay showing mock edited cells versus cells edited with each CD4 expression cassette. Bars represent relative frequency of each progenitor colony type: CFU-GEMM (multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells), CFU-GM (colony forming unit-granulocytes and monocytes), BFU-E (erythroid burst forming units) (n=3 independent HSPC donors). Bars represent mean +/−SD.

Article Snippet: Briefly, isolated mononuclear cells were positively selected for CD34 using the CD34+ MicroBead Kit Ultrapure (Miltenyi Biotec, San Diego, CA, cat.: 130-100-453).

Techniques: Expressing, Plasmid Preparation, Colony-forming Unit Assay

Journal: bioRxiv

Article Title: Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

doi: 10.1101/2025.04.14.648845

Figure Lengend Snippet: A. Targeted integration of CD4-expression cassettes in CD34 + HSPCs at three different amounts of AAV6 per cell, 1250, 2500 and 5000 vg/cell. n=1. B. Total colony formation as a percentage of plated cells (500 cells plated per condition). Bars represent mean +/− SD. n=3 biological donors.. C. Total colony formation as a percentage of plated cells (500 cells plated per condition) (left) and colony distribution (right) of an edited HSPC donor edited with higher-quality, purified by cesium chloride (CsCl) gradient ultracentrifugation, AAV6. Bars represent relative frequency of each colony type: CFU-GEMM, CFU-GM, BFU-E. n=1 donor. All bars represent mean +/− SD.

Article Snippet: Briefly, isolated mononuclear cells were positively selected for CD34 using the CD34+ MicroBead Kit Ultrapure (Miltenyi Biotec, San Diego, CA, cat.: 130-100-453).

Techniques: Expressing, Purification

Journal: bioRxiv

Article Title: Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

doi: 10.1101/2025.04.14.648845

Figure Lengend Snippet: A. Diagram showing workflow of genome-editing and subsequent RBC differentiation of HSPCs. B. Percentage of CD71 + cells of CD34 - CD45 - cells on day 14 of RBC differentiation. n=8 for Mock, CD4-GPA, CD4-GPA-tEPOR, n=3 for CD4 and CD4-tEPOR. C. Percentage of CD4 + cells of live single cells on each day of RBC differentiation. n=6 for Mock, n=4 CD4-GPA, CD4-GPA-tEPOR, n=3 on day 4-14 for CD4 and CD4-tEPOR on day 4-14 and n=2 on day 0. D. Median fluorescence intensity (MFI) of CD4 signal within CD4 + cells on day 14 of RBC differentiation. n=3 for all conditions. ns, p=0.5919 for CD4 vs. CD4-GPA, *p=0.0224 for CD4-tEPOR vs. CD4-GPA-tEPOR by unpaired two-tailed t -test. All bars represent median +/−95% confidence interval and all replicates represent independent HSPC donors.

Article Snippet: Briefly, isolated mononuclear cells were positively selected for CD34 using the CD34+ MicroBead Kit Ultrapure (Miltenyi Biotec, San Diego, CA, cat.: 130-100-453).

Techniques: Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

doi: 10.1101/2025.04.14.648845

Figure Lengend Snippet: A. Percentage GPA + CD71 + cells of CD34 - CD45 - cells on day 14 of RBC differentiation. Lines represent median +/− 95% confidence interval. n=3 independent biological donors. B. Flow cytometry plots of expression of Band3 in differentiated Mock, CD4-GPA, and CD4-GPA-tEPOR RBCs on day 14 and 18 of RBC differentiation. Plots shown as percent of live single CD71 + cells. n=1.

Article Snippet: Briefly, isolated mononuclear cells were positively selected for CD34 using the CD34+ MicroBead Kit Ultrapure (Miltenyi Biotec, San Diego, CA, cat.: 130-100-453).

Techniques: Flow Cytometry, Expressing

Journal: Cartilage

Article Title: In Vitro Effects of Cetylated Fatty Acids Mixture from Celadrin on Chondrogenesis and Inflammation with Impact on Osteoarthritis

doi: 10.1177/1947603518775798

Figure Lengend Snippet: Fluorescence microscopy images of untreated and cetylated fatty acids mixture from Celadrin-treated human adipose-derived stem cells (hADSCs) spheroids labeled with Sox-9, aggrecan, chondroitin sulfate, col2a, keratan sulfate, and syndecan-3 antibodies after 1, 2, and 3 weeks of in vitro chondrogenic induction.

Article Snippet: Cell Culture Models Adipose-Derived Stem Cells for Chondrogenic Process Investigation Human adipose-derived stem cells (hADSCs) (StemPro Human Adipose-Derived Stem Cells, ThermoFisher) were used in this study as the chondrogenic differentiation process model. After defrosting, the cells were propagated under standard culture conditions (37°C, humidified atmosphere and 5% CO 2 ) in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g glucose and supplemented with 1.5 g of NaHCO 3 , 1% antibiotic-antifungal solution, and 10% heat-inactivated fetal bovine serum (FBS).

Techniques: Fluorescence, Microscopy, Derivative Assay, Labeling, In Vitro

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A Schematic of the hematopoietic differentiation system. Following embryoid body setup, BMP4, Activin A, CHIR99021, VEGF, and hematopoietic cytokines were added sequentially to induce HE cell formation and EHT. Cells of interest were sorted at day 8 of the protocol. B Sorting strategy for obtaining pure HE, EHT, and HSC‐like cell populations. At day 8 of differentiation, representative plots show the level of CD34 + cells following magnetic bead enrichment, separation on the basis of CD43 expression and further gating on CXCR4 − CD73 − and CD90 + VEcad + for HE and EHT cells; and CD90 + CD38 − for HSC‐like cells. C, D Pseudotime analysis of EHT populations taking a G0 (C) or S/G2M (D) path and corresponding bar graphs showing abundance of populations. E scCoGAPS mapping of cord blood CD34 + cells (CB HSC) to the EHT dataset and violin plot showing pattern weights. F scCoGAPS mapping of the human CS 13 dorsal aorta dataset on the EHT dataset, with plot showing colocalization of populations. G Pie charts showing abundance of each cell type mapping from the human CS 13 dataset onto HE, EHT, or HSC‐like cell types, according to the scCoGAPS analysis in Fig .

Article Snippet: CD34 MicroBead Kit, human , Miltenyi Biotec , Cat# 130‐046‐703.

Techniques: Expressing

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A–C Sorted HE and EHT cells were subcultured for 6 days (representative of n = 5). The levels of CD43 and CD34 markers (A) and the levels of CD43, GPA, and CD45 markers (C) were assessed at subculture days 3 and 6. (B) Representative pictures of the wells were taken every day during HE and EHT subculture. Scale bars, 100 µm. D Expression of globin genes in HSC‐like cells assessed by scRNAseq.

Article Snippet: CD34 MicroBead Kit, human , Miltenyi Biotec , Cat# 130‐046‐703.

Techniques: Expressing

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A Pyruvate is transported into mitochondria via the mitochondrial pyruvate carrier (MPC, inhibitor: UK5099) and converted to acetyl‐coA by pyruvate dehydrogenase complex (PDH, inhibitor: 1‐AA). Pyruvate dehydrogenase kinases (PDKs, inhibitor: DCA) negatively regulate PDH activity. B–E FACS‐sorted HE cells were subcultured with or without UK5099 (10 µM) or DCA (3 mM). Subculture day 3 representative GPA/CD43 plots (B, D) and subculture day 6 representative CD45/CD43 plots (C, E) are shown (see Fig for corresponding bar graphs). F Ratio of CFU‐E to CFU‐G, M, GM colonies ± SEM relative to the control condition obtained from HE cells subcultured with the indicated compounds for 6 days, see Fig for percentages ( n = 5 biological replicates with 1–2 technical replicates each, paired t ‐test). G Fold change in the expression of HBE1 or HBG1‐2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non‐treated cells ( n = 3 biological replicates, paired t ‐test). H Percentages of CD45 + CD56 + ± SEM cells obtained following 35‐day co‐culture of 3‐day subcultured HE cells with OP9‐DL1 stroma. During the 3‐day subculture, HE cells were treated with the indicated compounds. ( n = 3 biological replicates, one‐way ANOVA test, see Fig for plots). I–K Pregnant mice were injected with UK5099 or DCA at E9.5 and fetal livers were analyzed at E14.5 by flow cytometry. FL, fetal liver. Levels of T and B cells (I) and LT‐HSCs (J) as percentages in fetal liver are shown for control ( n = 10 biological replicates), UK5099‐treated ( n = 14 biological replicates), and DCA‐treated ( n = 16 biological replicates) conditions (one‐way ANOVA test). (K) The ratio of BFU‐E to CFU‐GM colonies obtained from sorted LT‐HSCs is shown (see also data in Appendix Fig ) (one‐way ANOVA test). CFU, colony‐forming unit; BFU, burst‐forming unit; E, erythroid; M, macrophage; G, granulocyte. L–P HE cells co‐cultured with OP9‐DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice. Bone marrow (BM) and thymi were harvested on week 12. (L) The percentages ± SEM of human CD4 + CD8 + double‐positive thymocytes in huCD45 + cells from the thymus are shown (Control, n = 6 biological replicates; DCA, n = 7 biological replicates; unpaired t ‐tests). The percentages ± SEM of human B cells (M), CLPs (N) from the BM at week 12, as well as myeloid cells from PB at week 8 (O) and myeloid cells from BM at week 12 (P) in huCD45 + cells are shown (Control, n = 6 biological replicates; DCA, n = 7 biological replicates; unpaired t ‐tests). PB, peripheral blood. Data information: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 MicroBead Kit, human , Miltenyi Biotec , Cat# 130‐046‐703.

Techniques: Activity Assay, Control, Expressing, Co-Culture Assay, Injection, Flow Cytometry, Cell Culture, Irradiation

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 3 CD43 + /GPA + cell frequencies ± SEM relative to the controls for all populations are presented (HE, n = 5 biological replicates; EHT, n = 6 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). B FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 3 CD43 + GPA + cell frequencies ± SEM relative to the control are shown ( n = 4 biological replicates, paired t ‐test). C Fold change of expression of MPC1 and MPC2 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, unpaired t ‐tests). Untr, untransduced. D HE cells were transduced with shScrambled (shScr), shMPC1, shMPC2, or both the day after the sort and day 3 CD43 + /GPA + cell frequencies ± SEM relative to shScr are presented ( n = 4 biological replicates; one‐way ANOVA test). Untr, untransduced. E FACS‐sorted HE cells were stained with CTV and fluorescence was assessed by flow cytometry for GPA + cells at day 3 of subculture with or without UK5099 (10 µM). Representative of n = 3. F, G FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 6 CD43 + (F) and CD43 + CD45 + (G) cell frequencies ± SEM relative to the control for all populations are presented (HE, n = 7 biological replicates; EHT, n = 7 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). H FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 6 CD43 + and CD43 + CD45 + cell frequencies ± SEM relative to the control are shown ( n = 3 biological replicates, paired t ‐test). I, J FACS‐sorted HE cells were subcultured for 3 days with or without UK5099 (10 µM). CTV for HE‐derived CD45 + cells (I) and HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 7 biological replicates, paired t ‐test) (J) are shown. K–N FACS‐sorted HE and EHT cells were subcultured with or without DCA (3 mM). Subculture day 3 (K, n = 3 biological replicates) and day 6 (L, HE, n = 5 biological replicates; EHT, n = 4 biological replicates) CD43 + GPA + cell frequencies ± SEM relative to the controls are shown (paired t ‐test). (M) CTV for HE‐derived GPA + cells at subculture day 3 is shown. Representative of n = 3. (N) Subculture day 6 CD43 + CD45 + cell frequencies ± SEM relative to the controls for both populations are shown (HE, n = 5 biological replicates; EHT, n = 4 biological replicates; paired t ‐tests). O Fold change of expression of PDK1 , PDK2 , PDK3 , and PDK4 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, paired t ‐tests). P HE cells were transduced with shScrambled (shScr) or shPDK1, shPDK2, shPDK3, or shPDK4 the day after the sort and day 6 CD43 + /CD45 + cell frequencies ± SEM relative to shScr are presented ( n = 3 biological replicates; one‐way ANOVA tests). Q CTV for HE‐derived CD45 + cells are shown. Representative of n = 3. R HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 4 biological replicates, paired t ‐test) are shown. S EdU incorporation ± SEM into HE cells was assessed by flow cytometry after a 24‐h pulse at days 1 and 2 of subculture with or without UK5099 (10 µM) or DCA (3 mM) ( n = 3 biological replicates). T, U Percentages of CFU assay colony types ± SEM obtained from HE cells subcultured with the indicated compounds for 3 days (U) ( n = 3 biological replicates, two‐way ANOVA test) or 6 days (T) ( n = 5 biological replicates, two‐way ANOVA test). CFU, colony‐forming unit; E, erythroid; M, macrophage; G, granulocyte; GEMM, mixed. V EryD and EryP CFU‐Es obtained from HE cells subcultured with the indicated compounds for 3 days. Scale bars, 100 µm. W Fold change in the expression of HBA1‐2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non‐treated cells ( n = 3 biological replicates, paired t ‐test). X Plots showing percentages of CD45 + CD56 + cells obtained following 35‐day co‐culture of 3‐day subcultured HE cells with OP9‐DL1 stroma. During the 3‐day subculture, HE cells were treated with the indicated compounds. Data information: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD34 MicroBead Kit, human , Miltenyi Biotec , Cat# 130‐046‐703.

Techniques: Control, Expressing, shRNA, Transduction, Staining, Fluorescence, Flow Cytometry, Derivative Assay, Colony-forming Unit Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet:

Article Snippet: CD34 MicroBead Kit, human , Miltenyi Biotec , Cat# 130‐046‐703.

Techniques: Recombinant, Saline, Flow Cytometry, Gene Expression, RNA Sequencing, Software

Journal: Cell Communication and Signaling : CCS

Article Title: The FACT-targeted drug CBL0137 enhances the effects of rituximab to inhibit B-cell non-Hodgkin’s lymphoma tumor growth by promoting apoptosis and autophagy

doi: 10.1186/s12964-022-01031-x

Figure Lengend Snippet: CBL0137 inhibits B-NHL cell proliferation by inducing cell cycle arrest and promoting apoptosis. A Cell viability of B-NHL cells treated with various concentrations of CBL0137 for 24 h was measured by CCK-8 assay. B B-NHL cells were treated with 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM CBL0137, and 2.0 μM doxorubicin for 24 h, 48 h, and 72 h. The absorbance values at 450 nm were determined. C Cell proliferation was detected by colony formation assay after 24 h treatment with CBL0137 (1.0 μM). D The cell cycle was determined by flow cytometry. The percentages of cell cycle phases were shown in the bar chart. CBL0137 induced a significant increase in the proportion of B-NHL cells in S phase. E The expression of cyclins, CDKs, c-MYC, p21, and p53 was detected by western blotting in B-NHL cells treated with CBL0137. F Four B-NHL cells (SU-DHL-4, Farage, Raji, and Jeko-1) were treated with specified concentrations of CBL0137 for 24 h. Cell apoptosis was assessed by the Annexin V-FITC Apoptosis Detection Kit and flow cytometry. Representative results are shown on the left and statistical results are shown on the right. G B-NHL cells were treated with different concentrations of CBL0137 for 24 h and then were collected, and chromosomal DNA was isolated and purified using DNA Ladder Detection Kit according to manufacturer's instructions to explore the effect of CBL0137 on DNA fragmentation in B-NHL cells. H , I Representative proteins expression in four B-NHL cells treated with different concentrations of CBL0137 and vehicle control (DMSO) for 24 h to evaluate the status of apoptotic cascade and mitochondrial apoptosis. The data are representative and taken from one of three independent experiments. The data results are expressed as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t-test)

Article Snippet: For cell cycle analysis, B-NHL cells were treated with CBL0137 (0.5 μM, 1.0 μM, 1.5 μM, and 2.0 μM) for 24 h and stained with PI/RNase (KeyGEN BioTECH, China) for 30 min. An Annexin V-FITC/PI Apoptosis Kit from Vazyme was used for apoptosis analysis.

Techniques: CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot, Isolation, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: The FACT-targeted drug CBL0137 enhances the effects of rituximab to inhibit B-cell non-Hodgkin’s lymphoma tumor growth by promoting apoptosis and autophagy

doi: 10.1186/s12964-022-01031-x

Figure Lengend Snippet: CBL0137 induces protective autophagy in B-NHL cells. A Autophagosomes in SU-DHL-4, Raji, and Jeko-1 cells treated with or without 2.0 μM CBL0137 were observed by TEM. The arrow indicates the autophagosome. Scale bars = 2 μm. B The expression of GFP + /mRFP + (yellow) and GFP-/mRFP + (red) LC3 puncta was observed by fluorescence microscopy in B-NHL cells treated with or without 1.0 μM or 2.0 μM CBL0137 for 24 h. Representative images and quantitative analysis of fluorescent LC3 puncta were shown. Scale bars = 30 μm. C Expression of autophagy-related proteins was detected by western blotting in B-NHL cells treated with CBL0137 or DMSO. D – F Effect of chloroquine (CQ) on CBL0137-mediated autophagy, cell viability, and apoptosis. Cells were pretreated with 50 μM CQ for 1 h and then exposed to 1.0 μM CBL0137. D Cell viability was determined by CCK-8. E Apoptosis was measured by flow cytometry. F Conversions of LC3I to LC3II, p62, Atg14, and PARP were examined by western blotting. G B-NHL cells were pretreated with 2.5 mM 3-methyladenine (3-MA) for 1 h and then exposed to 2.0 μM CBL0137. The expression of PARP, p62, LC3, Bcl-xL was detected by western blotting. H 50 μM Z-VAD-FMK was pretreated for 1 h and then exposed to 2.0 μM CBL0137. Caspase-related proteins and autophagy-related proteins were detected by using western blotting

Article Snippet: For cell cycle analysis, B-NHL cells were treated with CBL0137 (0.5 μM, 1.0 μM, 1.5 μM, and 2.0 μM) for 24 h and stained with PI/RNase (KeyGEN BioTECH, China) for 30 min. An Annexin V-FITC/PI Apoptosis Kit from Vazyme was used for apoptosis analysis.

Techniques: Expressing, Fluorescence, Microscopy, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: The FACT-targeted drug CBL0137 enhances the effects of rituximab to inhibit B-cell non-Hodgkin’s lymphoma tumor growth by promoting apoptosis and autophagy

doi: 10.1186/s12964-022-01031-x

Figure Lengend Snippet: CBL0137-induced apoptosis and autophagy are dependent on mitochondrial ROS generation. A Jeko-1 cells treated with 1.0 μM or 2.0 μM CBL0137 were stained with the fluorescent mitochondrial probe JC-1, and the mitochondrial membrane potential (MMP) of the cells was observed by fluorescence microscope (40×, magnification). The quantitative results were analyzed. B The MMP stained with JC-1 probe was measured by flow cytometry. The histogram showed the percentage of JC-1 aggregate monomer. The data were expressed as mean ± SD (n = 3). C B-NHL cells were treated with different concentrations of CBL0137 for 24 h and then incubated with 10 μM DCFH-DA at 37 °C in the dark for 20 min, the fluorescent intensity was detected by flow cytometry. The mean fluorescence intensity of ROS was shown in histograms. The data were expressed as mean ± SD (n = 3). D B-NHL cells were preincubated with 2.5 mM NAC for 2 h and then treated with 2.0 μM CBL0137 for 24 h. The fluorescence intensity was detected by flow cytometry. E B-NHL cells were preincubated with 2.5 mM NAC for 2 h and then treated with 1.0 μM CBL0137 for 24 h. The changes in cell apoptosis rates were measured by flow cytometry. F In the presence or absence of NAC preincubation, the changes of apoptosis and autophagy-related proteins expression in B-NHL cells after CBL0137 treatment were detected by western blotting. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For cell cycle analysis, B-NHL cells were treated with CBL0137 (0.5 μM, 1.0 μM, 1.5 μM, and 2.0 μM) for 24 h and stained with PI/RNase (KeyGEN BioTECH, China) for 30 min. An Annexin V-FITC/PI Apoptosis Kit from Vazyme was used for apoptosis analysis.

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Incubation, Expressing, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: The FACT-targeted drug CBL0137 enhances the effects of rituximab to inhibit B-cell non-Hodgkin’s lymphoma tumor growth by promoting apoptosis and autophagy

doi: 10.1186/s12964-022-01031-x

Figure Lengend Snippet: Effect of the PI3K/Akt/mTOR and MAPK signaling pathways on CBL0137-induced apoptosis and autophagy. A Singal pathway enrichment analysis of CBL0137 2.0 μM treated and untreated groups by RNA-seq in SU-DHL-4, Raji, and Jeko-1 cells ( P < 0.05). B B-NHL cells were incubated with CBL0137 (0.5–2.0 μM) for 24 h, after which levels of PI3K, AKT, mTOR, and their phosphorylated forms were determined by western blotting. C Western blotting was used to detect the expression changes of related downstream molecules in the MAPK signaling pathway after CBL0137 (0.5–2.0 μM) treatment, including ERK1/2, p38 MAPK, p-p38 MAPK, GSK3β, and their phosphorylated forms. D B-NHL cells were pretreated with 10 μM U0126 or 10 μM LY294002 for 2 h followed by exposed to 2.0 μM CBL0137 for 24 h, respectively. Western blotting was used to detect the expression of apoptotic and autophagic marker proteins, and GAPDH was used as an internal control. E The expression level of cleaved PARP and the conversion of LC3 were analyzed by western blotting when 10 μM U0126 and 10 μM LY294002 were used together. F B-NHL cells were treated with 2.0 μM CBL0137 from 0 to 24 h. Western blotting was used to analyze the expression of key molecules involved in apoptosis and autophagy, as well as changes in the phosphorylation status of PI3K, Akt, mTOR, and ERK1/2. G B-NHL cells were treated with CBL0137 or in combination with NAC. The expression of p-PI3K, p-AKT, p-mTOR, and p-ERK1/2 was detected by western blotting in B-NHL cells

Article Snippet: For cell cycle analysis, B-NHL cells were treated with CBL0137 (0.5 μM, 1.0 μM, 1.5 μM, and 2.0 μM) for 24 h and stained with PI/RNase (KeyGEN BioTECH, China) for 30 min. An Annexin V-FITC/PI Apoptosis Kit from Vazyme was used for apoptosis analysis.

Techniques: RNA Sequencing Assay, Incubation, Western Blot, Expressing, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: The FACT-targeted drug CBL0137 enhances the effects of rituximab to inhibit B-cell non-Hodgkin’s lymphoma tumor growth by promoting apoptosis and autophagy

doi: 10.1186/s12964-022-01031-x

Figure Lengend Snippet: The combination of CBL0137 and rituximab exerted enhanced effects on B-NHL tumors in vitro and in vivo. A The synergy scores of CBL0137 and rituximab were calculated using the Bliss analysis. The concentration gradients of CBL0137 were 0.5, 1.0, 1.5, and 2.0 μM, and the concentration gradients of rituximab were 10, 20, 30, and 40 μg/mL. The combination responses of the two drugs were observed through the dose–response matrix. The 2D and 3D synergy maps above and below highlighted synergistic and antagonistic dose regions in red and green color respectively and showed the summary synergy scores of two drug combinations in three B-NHL cell lines. B LDH release assay of B-NHL cells treated with the indicated concentration of CBL0137 or rituximab and the combination of the two drugs for 24 h. C The Annexin V-FITC/PI staining results were shown on the left, which was that CBL0137 combined with rituximab has enhanced apoptotic effects on SU-DHL-4 and Raji cells. Statistical results of apoptosis assay were shown on the right, representing three independent experiments. (* P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t-test). D , E Representative western blotting results of B-NHL cells treated with the single or combination treatment for 24 h to determine changes in apoptosis-related proteins D and autophagy-related proteins E expression. F Experimental protocol of xenograft model of DLBCL. G SU-DHL-4 cells were subcutaneously implanted into male nude mice, and the mice were sacrificed after the treatment. The mice and their corresponding xenograft tumor specimens were photographed with a high-definition digital camera. H Tumor volume was measured every three days and tumor growth curves were plotted. *** P < 0.001, **** P < 0.0001. I Tumor weight in each group was measured and presented at the end of treatment. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J The body weight of mice in four groups (n = 8) was recorded weekly. K Histological examination of vital organs was performed by H&E staining. Scale bars = 50 μm. L Representative immunohistochemical images of Ki67, cleaved caspase-3, and LC3B were performed in the four groups of tumor samples. Scale bars = 50 μm. M , N Establishment of BL xenograft tumors. Raji cells were subcutaneously implanted into female nude mice, and the mice were sacrificed after the treatment. M Quantified results of tumor volume. N Quantified results of tumor weight

Article Snippet: For cell cycle analysis, B-NHL cells were treated with CBL0137 (0.5 μM, 1.0 μM, 1.5 μM, and 2.0 μM) for 24 h and stained with PI/RNase (KeyGEN BioTECH, China) for 30 min. An Annexin V-FITC/PI Apoptosis Kit from Vazyme was used for apoptosis analysis.

Techniques: In Vitro, In Vivo, Concentration Assay, Lactate Dehydrogenase Assay, Staining, Apoptosis Assay, Western Blot, Expressing, Immunohistochemical staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HCK maintains the self-renewal of leukaemia stem cells via CDK6 in AML

doi: 10.1186/s13046-021-02007-4

Figure Lengend Snippet: HCK is required for leukaemogenesis and self-renewal of LSCs. (a-c) Genome PCR, RT-PCR, and western blotting were performed with HCK −/− BM cells to confirm HCK deletion. NC: Negative control; PC: Positive control. (d) Experimental scheme for investigating the function of HCK in leukaemogenesis. (e) Frequency of YFP + leukaemia cells in the peripheral blood at 32 days after the second transplantation. (f) Representative images of the sizes of spleens of recipient mice upon the second transplantation. (g) Quantification of the weights of the spleens in f. (h) Histological H&E staining of the spleens in f. (i) Representative images of colonies formed by YFP + c-Kit + LSCs from secondary recipients. (j) The colony numbers were calculated in i. (k) Survival data for recipient mice receiving HCK −/− or HCK +/+ YFP + c-Kit + LSCs upon the second transplantation ( n = 10; log-rank test). (l) Frequency of YFP + leukaemia cells in the peripheral blood at 32 days after the third transplantation. (m) Survival data for recipient mice receiving HCK −/− and HCK +/+ YFP + c-Kit + LSCs upon the third transplantation ( n = 5; log-rank test). (n) Representative images of colonies formed by YFP + c-Kit + LSCs from the third transplant recipient. (o) The colony numbers were calculated in n. (p) Representative FACS plot and graph of c-Kit and Gr1 expression in HCK −/− and HCK +/+ leukaemic cells to assess the frequency of LSC. (q) Cell cycle status was determined in the third transplant recipient in vivo. (r) LSC frequency was determined from a limiting dilution assay performed with BM cells from the third transplant recipient mice. The ELDA web tool was used to calculate the frequency of LSCs

Article Snippet: A colony assay for murine LSCs was performed by plating 500 sorted YFP + c-Kit + LSCs on methylcellulose (MethoCult M3434, Stem Cell Technologies), and colonies were counted 14 days later.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control, Positive Control, Transplantation Assay, Staining, Expressing, In Vivo, Limiting Dilution Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HCK maintains the self-renewal of leukaemia stem cells via CDK6 in AML

doi: 10.1186/s13046-021-02007-4

Figure Lengend Snippet: HCK is required for AML maintenance. (a) Experimental scheme for investigating the function of HCK in AML maintenance. (b) PCR-based confirmation of HCK deletion in MLL-AF9 leukaemia cells following tamoxifen delivery in vivo. (c) Frequency of YFP + leukaemia cells in the peripheral blood at the indicated time after the second transplantation (n = 10). (d) Representative images of the sizes of spleens of recipient mice. (e) Quantification of the weights of the spleens in d. (f) Histological H&E staining of the spleens in d. (g-h) Representative images and number of colonies formed by YFP + c-Kit + LSCs from the secondary recipients. (i) Survival data upon the secondary recipient mice (n = 10; log-rank test). (j) Representative FACS plot and graph of c-Kit and Gr1 expression in HCK −/− and HCK +/+ MLL-AF9 leukaemic cells to assess the frequency of LSCs. (k) Representative flow cytometric analysis of the cell cycle distribution in HCK −/− and HCK +/+ leukaemic cells from the second transplant recipient in vitro

Article Snippet: A colony assay for murine LSCs was performed by plating 500 sorted YFP + c-Kit + LSCs on methylcellulose (MethoCult M3434, Stem Cell Technologies), and colonies were counted 14 days later.

Techniques: In Vivo, Transplantation Assay, Staining, Expressing, In Vitro