Journal: PLoS ONE

Article Title: Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α

doi: 10.1371/journal.pone.0130118

Figure Lengend Snippet: A) Representative micrographs of suspension colonies from parental (ADH) and CD133 + cells. B) Semisolid colony-forming cell (CFC) assay. Parental (ADH) and prostasphere (SUS) cultures were dissociated at day 12 of culture. CD133 + cells were isolated via MACS using the 293C3 antibody. 1x10 3 cells from each condition were seeded in 35-mm petri dishes with StemXVivo methylcellulose concentrate mixed with DMEM-FBS or hPCM-PLUS. Results are expressed as means ± StdDev. (* p < 0.05). C) Representative micrographs of adherent colonies from parental (ADH) and CD133 + cells. D) Adherent colony-forming cell (CFC) assay. Cells were processed as described before for semisolid CFC assay. Then, 1x10 3 cells were seeded in 35-mm petri dishes with DMEM-FBS or hPCM-PLUS. Results are expressed as means ± StdDev. (* p < 0.05).

Article Snippet: Suspension CFC assays were carried out using StemXVivo methylcellulose concentrate (R&D Systems) combined with DMEM-FBS without phenol red or hPCM-PLUS media.

Techniques: Suspension, Hematopoietic Colony Assay, Isolation

Journal: Stem cells (Dayton, Ohio)

Article Title: Age-dependent depletion of human skin-derived progenitor cells.

doi: 10.1002/stem.27

Figure Lengend Snippet: Figure 2. In vitro passage of human skin-derived precursor (SKP) cells is de- pendent on expansion protocol and donor age. (A): Adult human dermospheres were passaged in liquid proliferation medium (left panel) or semisolid, methylcellulose- containing medium (right panel) for four passages. Total dermosphere number at each passage was calculated for a seeding cell density of 10,000 cells per square cen- timeter. Two independent donor biopsies were used for each experiment. Adult human SKPs were only expanded initially in liquid proliferation medium and in a more prolonged manner when semisolid medium was used. (B): Expansion of human infant and newborn (postnatal day 3) rat dermospheres in liquid proliferation medium. Two independent human or rat SKP cultures were passaged at a constant density of 10,000 cells per square centime- ter and the resulting dermospheres quanti- fied at day 7 in proliferation medium. Both infant human cells and rat SKPs were seri- ally passaged as dermosphere cultures.

Article Snippet: For methylcellulose passaging experiments, methylcellulose stock solution (2.8%, R&D Systems, Minneapolis, MN, http://www.rndsystems.com HSC001) was diluted to a final concentration of 0.8% in proliferation medium.

Techniques: In Vitro, Derivative Assay

Journal: Stem Cell Reviews and Reports

Article Title: Extracellular Adenosine (eAdo) - A 2B Receptor Axis Inhibits in Nlrp3 Inflammasome-dependent Manner Trafficking of Hematopoietic Stem/progenitor Cells

doi: 10.1007/s12015-022-10417-w

Figure Lengend Snippet: Expression of P1 receptors on murine and human HSPCs and their chemotactic responsiveness. Expression levels of Adora A 1 , A 2A , A 2B and A 3 receptors mRNA ( Panel A ) were evaluated by qRT-PCR analysis in human UCB (left) and murine BM derived (right) MNCs and HSPCs. The expression was normalized to β-2microglobuline Ct value and shown relative to A 1 receptor mRNA. The data are presented as means ± SD from 3 separate analysis. Samples were separated on 2% agarose gels stained with ethidium bromide to visualize qRT-PCR reaction products. Representative image is shown. ( Panel B ) Transwell migration assay (pore size 5 μm) with WT murine BMMNC (left panel) and human UCBMNC (right panel). Data shows the number of CFU-GM colonies migrating to ATP (0.25 µg/ml) alone or in the presence of A 2A inhibitor (SCH442416, 10 µM) or A 2B inhibitor (PSB1115, 10 µM). Number of CFU-GM colonies was assayed in methylcellulose cultures after plating cells that migrated to lower Transwell chambers. The data are presented as means ± SE. Unpaired Student’s t -test was used for the determination of significance (*p ≤ 0.05 and #p ≤ 0.005)

Article Snippet: The rest of the BMMNCs recovered from the lower chamber were resuspended in a human methylcellulose base medium provided by the manufacturer (R&D Systems), supplemented with murine GM-CSF (25 ng/ml) and IL-3 (10 ng/ml) for determining the number of CFU-GM colonies.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Staining, Transwell Migration Assay, Pore Size

Journal: Nature Chemical Biology

Article Title: New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

doi: 10.1038/nchembio.1930

Figure Lengend Snippet: Figure 1 | Identification of novel allosteric inhibitors of mutant IDH1. (a) Chemical structures of GSK321, an active IDH1 mutant inhibitor, and GSK990, a structurally related inactive inhibitor. (b) Potent inhibition of mutant IDH1 enzyme leads to decreased production of intracellular 2-HG in IDH1 R132C mutant HT-1080 cells after treatment with GSK321 for 24 h. No inhibition is seen after treatment with GSK990. Half-maximal effective concentration (EC50) values for decreased intracellular 2-HG levels are indicated. Graphs are representative of 2–8 independent experiments. (c) GSK321 selectively interacts with IDH1. A GSK321 derivative was immobilized to Sepharose beads and used to pull down cellular target proteins from HT-1080 cell lysate. Mass spectrometry was used to identify peptides from proteins bound by the IDH1 inhibitor matrix. A comparison of peptide enrichment observed in the presence of vehicle or competing compound demonstrated that GSK321 uniquely competes IDH1 from the matrix. (d) Dose- response curve showing GSK321 binding affinity for IDH1 in HT-1080 cell lysate. Binding of IDH1 to the IDH1 inhibitor matrix was assessed in the presence of increasing concentrations of free GSK321 to determine the percent inhibition. Mean of two experimental replicates is shown. (e) GSK321 inhibits 2-HG production within 24 h and is rapidly reversible upon compound washout. Data presented as mean ± s.e.m. (N = 4).

Article Snippet: 1.5 × 105 to 5 × 105 IDH1 mutant AML cells per mL were plated in HSC003 methylcellulose media containing IL-3, SCF, GM-CSF, and EPO (R&D Systems), and Primocin in the presence of DMSO (0.3%), 3 μM GSK321 or GSK990 IDH1 inhibitors.

Techniques: Mutagenesis, Inhibition, Concentration Assay, Mass Spectrometry, Comparison, Binding Assay

Journal: Nature Chemical Biology

Article Title: New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

doi: 10.1038/nchembio.1930

Figure Lengend Snippet: Figure 2 | Structural and biochemical characterization. (a) Crystal structure of GSK321 bound to the R132H IDH1 homodimer. GSK321 (yellow) is bound in an allosteric pocket in both monomers of the IDH1 R132H dimer. NADP+ (magenta) and His132 are also shown. (b) Detailed view of the allosteric binding pocket for GSK321. GSK321 (yellow) is bound mainly through hydrogen bonds to the backbone of IDH1 (green). (c) Overlay of one monomer of the IDH1 R132H–NADP+ binary complex (open form, green) bound to GSK321 (yellow) and the IDH1 R132H–NADP+–Ca2+–αKG ternary complex (closed form, gray). The inhibitor is wedged next to Seg-2, preventing its full organization into the active enzyme conformation. (d,e) Biochemical MOI of GSK849. Competitive inhibition is observed between GSK849 and αKG (Vmax = 20 ± 1 min−1; KαKG = 2.2 ± 0.4 mM; Ki = 31 ± 5.2 nM), whereas mixed/noncompetitive inhibition is observed between GSK849 and NADPH (Vmax = 62 ± 1.5 min−1; KNADPH = 1.0 ± 0.10 μM; GSK849 Kis = 205 ± 102 nM and GSK Kii = 70 ± 5.0 nM). Averages of two independent experiments are shown. N = 2 independent repeats. (f) Thermal stabilization data for GSK849 and GSK321. Binding was observed with either cofactor-free or NADPH-saturated enzyme (mean and s.d. are shown for a total of N = 6 replicates). (g) GSK321, but not GSK990, leads to reduction of histone H3K9 dimethylation (H3K9me2). Representative gel depicted of N = 6 total replicates. R132C IDH1–expressing HT-1080 cells were treated for 48 h with either GSK321 or GSK990. Total H3 and β-actin are shown as loading controls (see full gel images in Supplementary Fig. 1b).

Article Snippet: 1.5 × 105 to 5 × 105 IDH1 mutant AML cells per mL were plated in HSC003 methylcellulose media containing IL-3, SCF, GM-CSF, and EPO (R&D Systems), and Primocin in the presence of DMSO (0.3%), 3 μM GSK321 or GSK990 IDH1 inhibitors.

Techniques: Binding Assay, Inhibition, Expressing

Journal: Nature Chemical Biology

Article Title: New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

doi: 10.1038/nchembio.1930

Figure Lengend Snippet: Figure 3 | GSK321 decreases intracellular 2-HG and affects proliferation of primary IDH1 mutant AML cells. (a) Intracellular 2-HG levels following the treatment of IDH1 mutant AML cells with GSK321 or GSK990 as compared to that in DMSO-treated cells. Shown are mean values for two biological replicates. 2-HG levels are normalized to total cell numbers and expressed as fold change relative to the DMSO control. (b) Transient increase in absolute cell numbers after treatment of IDH1 mutant AML cells with GSK321. Cell numbers are displayed relative to those for DMSO-treated cells (dotted lines). Error bars, s.e.m. (N = 3 independent biological samples each for R132G, R132C and R132H, and N = 2 WT IDH1 biological AML samples in technical duplicates (total N = 4); N = 2 technical replicates for healthy BM WT IDH1.) P-values were determined using two-way ANOVA. (c) Representative FACS plots of cell cycle analysis of R132G IDH1 AML cells after treatment in suspension culture for 7 and 15 d, respectively. (d) Quantification of cell cycle distribution. Hoechst and pyronin-Y staining were performed. Total N = 3 for two independent biological samples each for R132G and R132C. P-values were calculated using two-way ANOVA. (e) Evaluation of cell death by annexin-V staining of cells from individuals with AML with mutant IDH1, treated for the indicated time points. Representative FACS plots are depicted for each mutant. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: 1.5 × 105 to 5 × 105 IDH1 mutant AML cells per mL were plated in HSC003 methylcellulose media containing IL-3, SCF, GM-CSF, and EPO (R&D Systems), and Primocin in the presence of DMSO (0.3%), 3 μM GSK321 or GSK990 IDH1 inhibitors.

Techniques: Mutagenesis, Control, Cell Cycle Assay, Suspension, Staining

Journal: Nature Chemical Biology

Article Title: New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

doi: 10.1038/nchembio.1930

Figure Lengend Snippet: Figure 4 | Induction of differentiation in primary IDH1 mutant AML blasts and immature stem-like cells. (a) Cytospins showing signs of granulocytic maturation (black arrows) after 9 d of treatment. Scale bars, 20 μm. (b) Quantification of myeloid differentiation after 9 d of treatment of R132G IDH1 mutant AML (box, 25th–75th percentiles; whiskers, minimum and maximum values; P-value determined by one-way ANOVA Friedman test). (c) FACS plots of CD15 expression after 7 d of treatment. (d) Increase in the number of CD15+ cells in IDH1 mutant AML cells after GSK321 treatment compared to DMSO- or GSK990-treated controls; expressed as fold change with respect to DMSO-treated group after 3–4 and 6–7 d, respectively (N = 3 R132G, N = 2 R132C and N = 3 R132H independent biological samples). (e) Representative CD45/SSC FACS dot plots with three gates (G/M, granulocytes/monocytes (SSChighCD45+); L, lymphocytes (SSClowCD45high); B, blasts (SSClowCD45low/+) after 7 d of treatment. (f) Quantification of cell distribution in blast, G/M and L gates as displayed in e. Each dot represents an independent biological sample (N = 4) (P-value determined using two-way ANOVA). (g) Histogram plots of CD15 expression on cells within the blast and G/M gates in e. (h) FACS analysis of immature hematopoietic stem cell (HSC) and progenitor (Prog.) cell compartments. Error bars, s.d. (N = 3 independent WT IDH1 biological samples). (i) Representative FACS plots of CD38 expression within lineage-negative (Linneg) cells, after 5 d of treatment. (j) Percentages of CD38-expressing cells within the Linneg gate are quantified as fold change relative to DMSO (unpaired Student′s t-test). Each dot represents an independent biological sample (N = 3 WT AML and N = 7 IDH1 mutant AML). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: 1.5 × 105 to 5 × 105 IDH1 mutant AML cells per mL were plated in HSC003 methylcellulose media containing IL-3, SCF, GM-CSF, and EPO (R&D Systems), and Primocin in the presence of DMSO (0.3%), 3 μM GSK321 or GSK990 IDH1 inhibitors.

Techniques: Mutagenesis, Expressing

Journal: Nature Chemical Biology

Article Title: New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

doi: 10.1038/nchembio.1930

Figure Lengend Snippet: Figure 5 | GSK321 leads to genome-wide DNA cytosine hypomethylation in IDH1 mutant AML cells. IDH1 R132G AML cells were treated in suspension culture with controls (DMSO or 3 μM GSK990) or 3 μM GSK321 for 6 d. (a) Volcano plot depicting differentially methylated CpGs. (b) Stacking bar plots showing the percentage of hypermethylated (magenta) and hypomethylated CpGs (green) out of all covered CpGs for each somatic chromosome after GSK321 inhibitor treatment compared to those of controls (DMSO-treated, GSK990-treated). (c) Pie chart illustrating all assayed methylation sites and the proportion of differentially methylated cytosines (DMCs) annotated to (epi)genomic features upon treatment with GSK321 (CpG islands, green; CpG shores, gray; and non–CGI-related regions, white). (d) Pie chart illustrating all assayed methylation sites and the proportion of differentially methylated cytosines (DMCs) upon GSK321 treatment annotated to promoter regions (red), exons (green), introns (blue) and intergenic regions (violet) throughout the genome. (e) Venn diagram representing the overlap between hypomethylated annotated CpGs (7,339) in GSK321-treated IDH1 mutant AML cells and hypermethylated CpGs (784) in IDH1 knock-in (KI) KSL (c-Kit+Sca1+Lin−) and human IDH1 or IDH2 mutant AML7. Out of these 784 genes hypermethylated in IDH mutant AML cells, 546 were hypomethylated upon treatment with the active IDH1 inhibitor. P-value < 0.0001.

Article Snippet: 1.5 × 105 to 5 × 105 IDH1 mutant AML cells per mL were plated in HSC003 methylcellulose media containing IL-3, SCF, GM-CSF, and EPO (R&D Systems), and Primocin in the presence of DMSO (0.3%), 3 μM GSK321 or GSK990 IDH1 inhibitors.

Techniques: Genome Wide, Mutagenesis, Suspension, Methylation, Knock-In

Journal: Advanced Healthcare Materials

Article Title: Suppressing t(4;11) Acute Leukemia by Lipopolymer Nanoparticle Delivery of siRNA Targeting KMT2A::AFF1 with Enhanced Extrahepatic Delivery

doi: 10.1002/adhm.202502019

Figure Lengend Snippet: Anti‐Leukemic Effects of siKMAF LPNP Treatment Alone and in Combination with Venetoclax in t (4;11) Acute Leukemia Cells. RS4;11 and SEM cells were treated with either scrambled siRNA (CsiRNA) or KMT2A::AFF1 ‐specific siRNA (siKMAF), with or without Venetoclax ( n = 3). After 24 h, cells were transferred to methylcellulose, and colony formation was assessed on day 14 by counting colonies under a microscope. A) Representative images of colonies from each treatment group in RS4;11, and SEM cells. B) Quantification of colony formation in RS4;11 cells: (Bi) total colony count and (Bii, Biii) colonies derived from specific hematopoietic progenitors. C) Quantification of colony formation in SEM cells: (Ci) total colony count and (Cii, Ciii) colonies derived from specific hematopoietic progenitors. Data are presented as mean ± SD. Statistical significance was determined using an unpaired t ‐test. UT: Untreated cells; Vent: Venetoclax; BFU‐E: Burst‐forming unit‐erythroid; CFU‐GM: Colony‐forming unit‐granulocyte, macrophage.

Article Snippet: A total of 1500 RS4;11 cells and 1000 SEM cells were seeded in 500 μL of human methylcellulose base media (Cat. #HSC005, R&D Systems, MN, USA), and the mixture was transferred into the inner wells of a 24‐well plate.

Techniques: Microscopy, Derivative Assay