Fig 1: Ephrin-A5 nanocaliper production workflow. (A) Schematic representation of the conjugation of ephrin-A5-Fc-His to an azide-oligo using bis-sulfone-PEG4-DBCO as a crosslinker. (B) Native-PAGE of ephrin-A5-oligo conjugates hybridized to complementary Alexa Fluor 647-labelled oligos, visualized by fluorescence imaging followed by silver staining. Bands corresponding to ephrin-A5-oligo conjugates (black bracket) before (BP) and after purification (AP) were visualized by fluorescence imaging (Cy5 channel) and by silver staining. Unconjugated ephrin-A5 (U) and azide-oligos (AO) mixed with Alexa Fluor 647-oligos were used as controls to check for presence of unconjugated protein (cyan arrowhead) and free azide-oligos (green arrowhead) respectively. Another control containing only the Alexa Fluor 647-oligo (CO) was run to check for free fluorophore-oligos (purple arrowhead). (C) Folding of single-stranded circular phage DNA (scaffold) with short ssDNA oligos (staples) designed to complementarily bind to different regions of scaffold to create the DNA origami nanocaliper structure. (D) An example of a DNA nanocaliper functionalized with ephrin-A5-oligo conjugates spaced 42.9 nm apart. (E) Nanocalipers not functionalized with protein (NC-empty) as well as nanocalipers functionalized with ephrin-A5 at different positions (NC-0, NC-14, NC-40 and NC-100), p7560 scaffold DNA (ssDNA scaffold) and 1 kb Plus DNA ladder were analyzed using a 2% agarose gel stained with ethidium bromide (left). The gel image is a composite of different lanes within the same gel, with the dotted lines delineating each part of the composition. Gel migration distances of different ephrin-A5 nanocalipers normalized to NC-empty (right). The number of ephrin-A5 dimers attached to the nanocalipers was consistent with the step-wise migration shift observed (n = 32 (NC-empty), n = 29 (NC-0), n = 18 (NC-14), n = 28 (NC-40), n = 18 (NC-100)). (F). Cells were stimulated by the addition of ephrin-A5 nanocalipers and analyzed with PLA, qRT-PCR and RNA-seq.
Fig 2: EphA2 receptor activation by ephrin-A5 nanocalipers in MDA-MB-231 and U3013 cell lines. (A) Quantification of EphA2 phosphorylation (pTyr) in MDA-MB-231 cells and (C) U3013 cells after 15- or 30-min stimulation with NC-empty, NC-0, NC-14, NC-40 and NC-100. P values indicated as * <0.05, ** <0.01, ***<0.001. Significance was determined by performing one-way ANOVA followed by Tukey HSD test. Error bars represent the standard error of the mean. Outliers were removed based on violations of normality and homogeneity of variances assumptions of ANOVA as determined by residuals analysis. Each dot represents the mean of each independent experiment containing 10–20 cells. (B) Representative PLA (EphA2-pTyr), DAPI (nucleus) and phalloidin (actin) microscope images of MDA-MB-231 cells and (D) U3013 cells. Scale bar denotes 50 μm.
Fig 3: Differential significantly enriched pathways in U3013 cells stimulated with ephrin-A5 nanocalipers. (A) Hypergeometric probability analysis of eight selected significantly enriched pathways in at least one data set. The dotted line signifies a P-value of 0.05. (B) A subset of genes in NC-100 data set which do not overlap with other nanocaliper data sets from the GSEA. (C) A subset of the spliceosome network of the DE genes of each nanocaliper data set overlaid with their respective –log2(FC) (colored).
Fig 4: Comparison of significantly enriched pathways between spatially controlled and uncontrolled-EphA2 stimulation. Euler diagram of significantly enriched pathways of ephrin-A5 nanocaliper data sets and IgG-clustered ephrin-A5.
Fig 5: U3013 cell transcriptome upon stimulation with ephrin-A5 nanocalipers. (A) (i) Circos plot depicting the fold changes of DE genes in cells stimulated with NC-0 (outer rim, dark orange), NC-14 (outer rim, green), NC-40 (outer rim, teal) and NC-100 (outer rim, orange). The size of each segment reflects the number of DE genes. The inner rims show the overlap of each data set with each other, each line representing one gene. The centre core shows the overall gene overlap between all data sets as represented by links. (ii–iv) The gene overlaps of one nanocaliper data set with the other data sets. ii. NC-0, iii. NC-14, iv. NC-40 and v. NC-100. Each gene overlap is represented by a link. (B) Correlation analysis of the complete nanocaliper data sets. The closer the data sets are to each other, the higher their correlation is. This is also represented by the thickness and the color of the line. The value next to each line corresponds to the correlation coefficient between two data sets. (C) Correlation analysis of the fold changes of gene overlaps between the nanocaliper data sets. Proportional Venn diagrams show the gene overlaps between the various data sets.
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