BMDM from AJ and B6 mice differ in their response to various stimuli.

<p>A) Stimulation of macrophages for ~ 18 hrs with IFNG+TNF resulted in high production of NO by AJ macrophages (black bars), while B) stimulation with LPS led to higher production of IL-12 in C57BL/6J (B6) macrophages (white bars). C) Stimulation of murine macrophages with LPS or CPG resulted in significantly higher production of IL-10 in B6 macrophages compared to the AJ macrophages. D) B6 macrophages stimulated with IL-4 produced significantly more urea, compared to AJ macrophages, while LPS stimulation induced low amounts of urea in both AJ and B6 BMDM. E) AJ macrophages stimulated with CPG or LPS produced significantly higher amounts of the chemokine CCL22 compared to B6 macrophages. F) The IFNG+TNF stimulated macrophages have increased toxoplasmacidal activity, which is slightly reduced in the presence of aminoguanidine (AG), an inducible nitric oxide synthase inhibitor. Where there was no detectable amount of cytokine/chemokine measured, including in all control (non-stimulated) BMDM, we do not include cytokine/chemokine data in the figures. Three independent replicates; Mean (SD) * <i>p < 0</i>.<i>05</i> (Student’s t-test).</p

QTL inheritance and allele effects on different traits in AXB/BXA BMDM.

<p>AA = Homozygous AJ allele; BB = Homozygous B6 allele. BMDMs were grouped based on their genotypes at the marker on the QTL peak. <i>P</i>-values were corrected for multiple testing (26 individuals) using the Bonferroni test.</p><p>QTL inheritance and allele effects on different traits in AXB/BXA BMDM.</p

shRNA- mediated <i>Ddx1</i> knockdown in C57BL/B6J immortalized macrophages relieves NO inhibition.

<p>Fold knockdown of <i>Ddx1</i> and fold change in NO levels are relative to the <i>Ddx</i>1 expression and the amount of NO in cells transduced with control shRNA (<i>LacZ</i>), respectively. Knocking down <i>E2f6</i>, the other candidate gene at this locus, did not affect the amount of NO produced. Shown are values of NO (μM) fold change obtained from two independent experiments using three different shRNA constructs. The knockdown level is indicated by the black triangles. Fold knockdown was calculated using the 2^deltadelta<i>Ct</i> method. The shRNA transductions and NO measurements were done in 3 independent replicates.</p

The transcriptional response in BMDM is regulated by stimulation-specific <i>trans</i>-loci.

<p>Expression quantitative trait loci (eQTL) in the RI mice were mapped in A) non-stimulated, B) IFNG+TNF-stimulated, C) <i>Toxoplasma</i>-infected, and D) CpG-Stimulated BMDM. Each dot represents a single eQTL (transcript). Significant eQTL located ≤ 10 Mb from the start of the physical location of the corresponding gene were designated as <i>cis</i> mapping (diagonal lines). All other eQTL were designated as <i>trans</i>-mapping (vertical lines). eQTL significance was calculated after 1000 permutations and reported at genome-wide thresholds corresponding to FDR ≤ 10%. Red spots identify genes mapping to a <i>trans</i>-eQTL hotpot (<i>trans</i>-band).</p

Functional enrichments in the large <i>trans</i>-eQTL hotspots in AXB/BXA macrophages following IFNG+TNF-stimulation, CpG-stimulation, or <i>Toxoplasma</i>-infection.

<p>Functional enrichments in the large <i>trans</i>-eQTL hotspots in AXB/BXA macrophages following IFNG+TNF-stimulation, CpG-stimulation, or <i>Toxoplasma</i>-infection.</p

Growth on patterned islands identifies factors that influence hiPSC-CM maturation.

<p>a) Cells were cultured on patterned islands with five regions in which different intercellular influences were active. b) Chart showing the modes of intercellular interaction active in each region. c) Image of hiPSC-CM cultured on patterned islands. Scale bar 500 μm. d) Amplitude of calcium transient; e) Amplitude of voltage transient; f) Action potential rise time, for the five regions indicated in (a). In (d-f) measurements were performed on <i>n</i> = 282 samples. Error bars represent s.e.m. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.</p

Optical electrophysiology recordings.

<p>(a) Fluorescence image of QuasAr2 expressed in 250 μm islands, all of which were monitored simultaneously at a 500 Hz frame rate. b) Fluorescence recordings of QuasAr2, indicating voltage, from islands in (a). The individual islands showed heterogeneity in spontaneous beat rate and action-potential waveform. c) Fluorescence of GCaMP6F expressed in the same islands as in (a). d) Fluorescence recordings of GCaMP6F, indicating Ca²⁺, from islands in (c). Data in (b) and (d) has been corrected for slow photobleaching, but not otherwise filtered.</p

Island size-dependent action potential parameters in hiPSC-CM.

<p>a) Mean spontaneous beat period as a function of island size at 14 dpp. b) Standard deviation of beat period across islands, as a function of island size. c) Amplitude of the calcium transient as a function of island size. d) Amplitude of the voltage transient as a function of island size. e) Rise time (20% to 80%) of the voltage transient as a function of island size. Error bars in a,c-e represent s.e.m.</p

Patterned cardiomyocytes.

<p>Cardiomyocytes were patterned on a cytophobic polyacrylamide surface patterned with covalently bound fibronectin by micro-contact printing. a) Trans-illumination image of patterned cardiomyocytes showing the full 6 mm field of view. b) Fluorescence images of cardiomyocytes on patterned islands with nuclear stain DAPI (blue) and myofibril stain cardiac troponin T (red). c) A magnified view of one 250 μm island. d) Myofibril banding is clearly visible at single-cell resolution.</p

RNA sequencing to probe the difference between hiPSC-CM grown in small (100 μm) islands vs. a confluent monolayer.

<p>a) Confluent cells had greater expression of cardiac-related genes, while cells grown on small islands had greater expression of genes related to extracellular matrix, adhesion, and angiogenesis. b) Gene expression differences between small islands and confluent monolayer, projected onto two vectors in gene-expression space. The CP → Adult axis comprises the 3,463 genes that were significantly differentially expressed between cardiac progenitors and adult cardiomyocytes. The Small Islands → Confluent monolayer axis comprises the 149 genes that were significantly differentially expressed between the two island sizes. ESC: embryonic stem cells MES: mesoderm, CP: cardiac progenitors, CM: embryonic stem cell-derived cardiomyocytes.</p