Extended Data Fig. 10: Effects of CCM2 and TLNRD1 knockdown relative to MAP3K3 knockdown and laminar flow.

a–d, CRISPRi TeloHAEC with control non-targeting guides or with guides to CCM2 or TLNRD1 (two guides apiece) were treated with 2 μg/ml doxycycline for 3 days and nucleofected with Cas9 particles containing control non-targeting guides or with 3 guides targeting exon 3 of MAP3K3 (Synthego). Cells were grown for 48 hours in media with 2 μg/ml doxycycline, and RNA harvested for bulk RNA-seq. The CCM2 guides reduced target gene expression, on average, by 3.4-3.6 fold (p. <2e-9), while TLNRD1 guides reduced target gene expression by 9.4-9.9 fold (p. <2e-43), consistent with the effects of these guides in our other bulk RNAseq data (Fig. 3c). MAP3K3 transcript levels were not significantly reduced, but genome-mappable reads for the targeted exon were greatly reduced, and most of the remaining reads showed multiple mismatches, indicating efficient introduction of Cas9-targeted deletions. N = 2 per condition (from one experiment, 1 RNAseq library for each of 2 CRISPRi guides per target). Correlation coefficient (R), and p-values given in each panel are from a two-sided Pearson correlation test. a, The difference between the effect of CCM2 knockdown in cells with MAP3K3 knockdown and the effect of CCM2 knockdown in control cells (log2 [CCM2kd_with_MAP3K3kd/Control_with_MAP3K3kd]/[CCM2kd/Control], Y-axis) was plotted against the effect of CCM2 knockdown in control cells (log2 [CCM2kd/Control], X-axis, plotting all genes regulated at p. <5e-4 in either contrast). Labelled genes are the top give up- or down-regulated genes by log2 fold change, on each axis. Diagonal line: slope -1 reference. b, As in a, but for the difference between the effect of TLNRD1 knockdown in cells with MAP3K3 knockdown and the effect of TLNRD1 knockdown in control cells (Y-axis), versus the effect of TLNRD1 knockdown in control cells (X axis). The negative correlations in a,b indicate that MAP3K3 perturbation partially reverses the transcriptomic effects of CCM2 or TLNRD1 knock down, consistent with a role of MEKK3/MAP3K3 signalling in regulating transcription downstream of both CCM2 & TLNRD1. c, As in a, but the difference between the effect of MAP3K3 knockdown in cells with CCM2 knockdown and the effect of MAP3K3 knockdown in control cells (Y-axis) versus the effect of MAP3K3 knockdown alone (X axis). d, As in a, but for the difference between the effect of MAP3K3 knockdown in TLNRD1 knockdown cells and the effect of MAP3K3 knockdown in control cells (Y-axis) versus the effect of MAP3K3 knockdown in control cells (X axis). The negative correlations in both c and d indicate that perturbation of CCM2 or TLNRD1 partially reverses the transcriptional effects of MAP3K3 knockdown, consistent with the expectation that decreased expression of upstream inhibitors can compensate for decreased expression of MEKK3. e–h, CRISPRi TeloHAEC with control non-targeting guides or with guides to CCM2 or TLNRD1 (2 guides apiece) were grown in static culture or subjected to flow in an Ibidi flow chamber for 48 h. In each case, cells were treated with 2 μg/ml doxycycline to induce the CRISPRi machinery for 5 days (3 days prior & 2 days after introduction of laminar flow). After phase contrast imaging, RNAseq libraries were prepared and sequenced to a depth of 10–12 million reads. N = 2 per condition (one experiment, with 1 RNAseq library for each of 2 CRISPRi guides per target). R and p-values, and labeled genes, as per a–d. e, The effects of CCM2 knockdown in static culture (Y-axis) compared to the effect of flow in control cells (X-axis, showing all genes regulated at p. <5e-4 in either contrast). Diagonal line: slope = +1 reference. f, As in e, but with Y-axis = effects of TLNRD1 knockdown in static culture. The positive correlations indicate that TLNRD1 or CCM2 knockdown in static culture is similar to the effect of flow. g, The difference between the effect of flow in cells with CCM2 knockdown and the effect of flow in control cells (log2 [Flow_CCM2kd/Static_CCM2kd]/[Flow_Ctrl/Static_Ctrl], Y-axis) versus the effect of flow in control cells (log2 [Flow_Ctrl/Static_Ctrl], X-axis). Diagonal line: slope = −1 reference. h, As in g, but showing the difference between the effect of flow in cells with TLNRD1 knock down and the effect of flow in control cells (Y-axis) versus the effect of flow in control cells (X-axis). The negative correlations in g,h indicate that CCM2 or TLNRD1 knockdown cells have a weaker transcriptional response to flow than control cells, consistent with these cells already having a partial flow-like transcriptional phenotype in static culture (e,f). i, Representative images of CRISPRi teloHAEC with the indicated guides, under laminar flow. N = 2 per condition from one experiment (two distinct CRIPSRi guides to CCM2, TLNRD1 or controls), and with four images per guide. j, The normal alignment to flow in control teloHAEC (measured as the angle, relative to flow, of the long axis of each cell) is significantly abrogated in both CCM2 & TLNRD1 KD cells (increased average angle relative to flow). Average values for all cells in each of four images for each of two guides per target were calculated (35 to 103 cells per image). Significance was assessed by a two sided T-test on these average values. N = 8. Boxplot features, as per Extended Data Fig. 1l. Note that alignment to flow is not completely blocked in CCM2 or TLNRD1 KD cells, since the average angle relative to flow does not reach the 45% value expected if orientation were entirely random. k, As per j, but measuring the ratio of the long versus short axis lengths for each cell (“length/width”) from the fit ellipse function in FiJi.