Figure 3: CDH2 knockdown (via CRISPRi) prevents cell cluster formation on soft, PEG-LM in porcine and human NP cells, with associated loss of juvenile NP phenotype. | Scientific Reports

Figure 3: CDH2 knockdown (via CRISPRi) prevents cell cluster formation on soft, PEG-LM in porcine and human NP cells, with associated loss of juvenile NP phenotype.

From: N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

Figure 3

(A) CRISPRi construct sequence. (B) Representative images of successful CDH2 knockdown analyzed by fluorescence microscopy. (C) Validation of successful CDH2 knockdown via flow cytometry. (D) Quantification of CDH2 knockdown via qRT-PCR; arrows indicate successful CDH2 knockdown using species-specific CRISPRs (porcine seq = porcine sequence, human seq = human sequence). (E) Representative immunostaining for phalloidin, CDH2 and β-catenin in porcine NP cells after CDH2 knockdown, compared to scramble control and no treatment (blue = protein, pink/red = cell nuclei, scale bar = 50 μm). (F) Quantification of total phosphorylated β-catenin levels in porcine NP cells (1 = CDH2 (−), 2 = scramble, 3 = no treatment). (G) Changes in sGAG in CDH2 knockdown porcine NP cells on soft. (H) Changes in gene expression for CDH2 knockdown porcine NP cells on soft, compared to stiff (CDH2 = N-cadherin, T = brachyury, LM1 = laminin, COL2 = type II collagen, AGC = aggrecan; * denotes that all genes are significantly different from scramble and no treatment controls) (I–L) same as (E–H) respectively, but with CDH2 knockdon in juvenile human NP cells (For all studies: 2-way ANOVA with Tukey’s post-hoc analysis, *p < 0.05).

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