Journal: Disease Models & Mechanisms

Article Title: Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

doi: 10.1242/dmm.052436

Figure Lengend Snippet: Characterisation of primary human peritoneal mesothelial cells (HPMCs) and human peritoneal fibroblasts (HPFs) compared to the LP-9 mesothelial cell line and normal human dermal fibroblasts (NHDFs). (A) Representative phase-contrast micrographs of LP-9, HPMCs, NHDFs and HPFs. Mesothelial and fibroblast cells exhibit cobblestone and spindle-like morphologies, respectively. (B) We observed localised expression of the cytoskeletal markers cytokeratin (CK) and vimentin (VIM) in LP-9 cells and HPMCs, while VIM, but not CK, was expressed in NHDFs and HPFs. HPMCs ( n =6) showed a high percentage of CK + /VIM + cells [98.20±1.05% (mean±s.d.)], while HPFs ( n =3) exhibited 81.36±5.63% CK − /VIM + cells. Scale bars: 200 µm (A); 50 µm (B).

Article Snippet: NHDFs were cultured in Fibroblast Growth Medium (Promocell, C-23110) containing 1 ng/ml recombinant human basic fibroblast growth factor and 5 μg/ml recombinant human insulin, supplemented with 2 mM L-glutamine and 100 μg/ml Primocin.

Techniques: Expressing

Journal: Disease Models & Mechanisms

Article Title: Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

doi: 10.1242/dmm.052436

Figure Lengend Snippet: Establishing composite 3D hydrogel constructs using peritoneal mesothelial cells and fibroblasts. (A) Schematic illustration of model construction and culture timeline. (B) Representative axial view [also seen in C (M3)] and Haematoxylin and Eosin (H&E)-stained section of a hydrogel construct showing the formation of a mesothelial monolayer (ML) and submesothelial layer (SML) on a transwell membrane (TM). (C) Representative images of hydrogel matrices using M1 (collagen I), M2 (70:30 collagen I:Matrigel ratio), M3 (50:50 collagen I:Matrigel ratio) and M4 (collagen I+human fibronectin). Construct generated with matrix combination M3 demonstrated minimal contraction in LP-9/NHDF and HPMC/HPF trials. (D) Lactate dehydrogenase (LDH) cytotoxicity assay in M3 composite hydrogel constructs containing HPMC/HPF ( n =3 donors) over a 10-day culture period. (E) Dual immunofluorescence staining of cleaved caspase-3 (CC-3) and VIM to detect apoptotic HPMCs/HPFs in M3 constructs on day 3 and day 10 of culture ( n =3). Scale bars: 300 µm (B); 100 µm (C); 50 µm (E).

Article Snippet: NHDFs were cultured in Fibroblast Growth Medium (Promocell, C-23110) containing 1 ng/ml recombinant human basic fibroblast growth factor and 5 μg/ml recombinant human insulin, supplemented with 2 mM L-glutamine and 100 μg/ml Primocin.

Techniques: Construct, Staining, Membrane, Generated, LDH Cytotoxicity Assay, Immunofluorescence

Journal: Disease Models & Mechanisms

Article Title: Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

doi: 10.1242/dmm.052436

Figure Lengend Snippet: Histological and functional analysis of the human parietal peritoneum and peritoneal layer models. (A) Histological staining of transverse sections through parietal peritoneum and composite 3D hydrogel constructs composed of LP-9/NHDFs and HPMCs/HPFs. Immunofluorescence using antibodies against the mesothelial markers podoplanin (PDPN) and mesothelin (MSLN), and submesothelial markers fibroblast specific protein 1 (FSP1) and tumor endothelial marker 1 (TEM1). (B) Colocalisation of MSLN and collagen IV (COLIV) suggesting spontaneous basal lamina formation. (C) Human tissue plasminogen activator (tPA) enzyme-linked immunosorbent assay (ELISA) to determine the functionality of the mesothelial cells in models assembled with HPMCs from three different donors over a 10-day culture period. Scale bars: 50 µm; 15 µm (insets in B).

Article Snippet: NHDFs were cultured in Fibroblast Growth Medium (Promocell, C-23110) containing 1 ng/ml recombinant human basic fibroblast growth factor and 5 μg/ml recombinant human insulin, supplemented with 2 mM L-glutamine and 100 μg/ml Primocin.

Techniques: Functional Assay, Staining, Construct, Immunofluorescence, Marker, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) In vivo CCS1477 treatment schematic for survival analyses. CD45.2 + D/F or T/F AML cells were transplanted into sublethally irradiated CD45.1 + WT recipient mice. Treatment was initiated 14 days posttransplant. CCS1477 (20 mg/kg) or vehicle was dosed by mouth (PO) once daily (QD). ( B ) Average ± SEM percent c-Kit + cells in the CD45.2 + peripheral blood (PB) of CCS1477- and vehicle-treated D/F ( n = 7 per treatment) and T/F ( n = 11 per treatment) transplanted AML mice over 2 weeks. Week 0 time point was immediately before the first treatment, followed by weeks 1 and 2 on treatment. Significant differences were evaluated by two-way analysis of variance (ANOVA) Šídák’s multiple comparisons test. Average ± SEM ( C ) spleen weight, and numbers of ( D ) CD45.2 + and ( E ) CD45.2 + LSK (Lin − Sca1 + Kit + ) bone marrow cells of D/F ( n = 8 per treatment) or T/F ( n = 8 per treatment) AML mice after 2 weeks of CCS1477 or vehicle. Significant differences were evaluated by unpaired t test. ( F ) Kaplan-Meier survival analysis of CCS1477-treated and vehicle-treated D/F AML (CCS1477, n = 12; vehicle, n = 14) and T/F AML (CCS1477, n = 9; vehicle, n = 9) transplant recipient mice. Overall survival (OS) of moribund D/F AML mice was 94 and 73 days for CCS1477 and vehicle, respectively. OS of moribund T/F AML mice was 51 and 39 days for CCS1477 and vehicle, respectively. Significance determined by the log-rank (Mantel-Cox) test. ( G ) Average ± SEM spleen weight at mouse moribundity of CCS1477- or vehicle-treated D/F ( n = 3 per treatment) or T/F ( n = 3 per treatment) AML mice. Significant differences were evaluated by unpaired t test. Individual data points represent biological replicates. For all panels, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For secondary transplants, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 700 rads before tail vein injection of 1 million c-Kit + AML cells from moribund D/F or T/F mice.

Techniques: In Vivo, Irradiation

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) Schematic of in vitro CCS1477 or vehicle treatments for H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). c-Kit + splenocytes from moribund D/F or T/F AML mice were treated for 1 or 6 hours before ChIP-seq or RNA-seq, respectively. ( B ) Quantification of log 2 fold change (adjusted p < 0.05) of H3K27ac at promoter and enhancer regions in CCS1477 versus vehicle in vitro–treated D/F ( n = 2 per treatment) and T/F ( n = 2 per treatment) c-Kit + AML cells after 1 hour. Statistical differences were evaluated by Wilcoxon test. ( C ) Heatmap of differentially expressed genes (cut-off log 2 fold change <−1, adjusted p < 0.05) by RNA-seq of c-Kit + AML D/F ( n = 3 per treatment) and T/F ( n = 3 per treatment) treated in vitro with CCS1477 or vehicle. ( D ) Heatmap depiction of gene set enrichment analyses (GSEA) pathways in common by H3K27Ac ChIP-seq and RNA-seq in CCS1477- versus vehicle-treated D/F (left) and T/F (right) AML cells. Color corresponds to GSEA normalized enrichment score (NES). ( E ) Representative H3K27ac ChIP-seq tracks at Myc blood enhancer cluster (BENC). ( F ) Average ± SEM Myc expression by RT-qPCR in CCS1477 versus vehicle in vitro–treated D/F ( n = 3 per treatment) and T/F ( n = 3 per treatment) c-Kit + AML cells after 6 hours. Significance determined by unpaired t tests. ( G ) Representative Western blot image for c-Myc in D/F and T/F AML cells after in vitro treatment of CCS1477 or vehicle after 6 hours. ( H ) Average ± SEM expression of apoptosis pathway genes by RT-qPCR in CCS1477- versus vehicle in vitro–treated D/F ( n = 4 per treatment) and T/F ( n = 4 per treatment) c-Kit + AML cells after 6 hours. Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. Individual data points represent biological replicates. For all panels, *** P < 0.001 and **** P < 0.0001.

Article Snippet: For secondary transplants, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 700 rads before tail vein injection of 1 million c-Kit + AML cells from moribund D/F or T/F mice.

Techniques: In Vitro, ChIP-sequencing, RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot

Journal: Science Advances

Article Title: P300/CBP inhibition with inobrodib in combination with gilteritinib and venetoclax targets leukemia stem cells in epigenetic mutant AML

doi: 10.1126/sciadv.aec9305

Figure Lengend Snippet: ( A ) Flow cytometric analyses of average ± SEM annexin V + cells 3 days after in vitro treatment of CCS1477, venetoclax, gilteritinib, combination, or vehicle in c-Kit + D/F AML cells ( n = 3). Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. ( B ) Average colonies [colony-forming units (CFU)] ± SEM formed by c-Kit + D/F AML cells ( n = 3) treated with CCS1477, venetoclax, gilteritinib, combination, or vehicle. Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. ( C ) Representative Western blot image for Mcl-1 in D/F AML cells after in vitro treatment with CCS1477, venetoclax, gilteritinib, combination, or vehicle after 4 hours in the presence of proteasome inhibitor. ( D ) Average ± SEM annexin V + cells by flow cytometric analyses after 3 days in vitro treatment of CCS1477, venetoclax, gilteritinib, combination, or vehicle in deidentified primary human FLT3 - ITD AML patient samples ( n = 10). Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. ( E ) Schematic of in vivo combination therapy. Mice received either CCS1477 ( n = 6), venetoclax ( n = 6), gilteritinib ( n = 6), combination ( n = 6), or vehicle ( n = 6) for 2 weeks. Mice were sacrificed 3 hours after the last treatment. Bar graphs of average ± SEM ( F ) spleen weight and ( G ) bone marrow CD45.2 + LSK (Lin − Sca1 + Kit + ) cells. Significance determined by one-way ANOVA with Tukey’s multiple comparisons test. Individual data points represent biological replicates. For all panels, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For secondary transplants, CD45.1 + C57BL6/J mice (Charles River Laboratories), 7 to 8 weeks of age, were irradiated with 700 rads before tail vein injection of 1 million c-Kit + AML cells from moribund D/F or T/F mice.

Techniques: In Vitro, Western Blot, In Vivo