Synaptopodin is required for α-actinin-4 recruitment to the cell junction. (A) Deconvolved optical section at the apical junction showing unchanged levels of E-cadherin, synaptopodin, and α-actinin-4 after prolonged cyclic pressure in mature cell monolayer. (B) Quantitation of junctional stainings in A before (U) and after (P) cyclic pressure. (C) Western blots showing upward shift of synaptopodin (black arrowheads) in a tension-sensitive manner. (D) Deconvolved optical section at the apical junction showing decreased α-actinin-4 and actin accumulation at the cell junction in synaptopodin knockdown cells. (E) Quantification of D before (U) and after (P) cyclic pressure. (B and E) Means are represented by horizontal lines. (F) Western blots showing α-actinin-4 level unchanged by knockdown of synaptopodin. (G) Wide-field images of venus-actinin showing cytoplasmic localization in synaptopodin knockdown cells. (H) BSA flux assays showing decreased barrier formation in synaptopodin knockdown monolayers. (I) Wide-field images showing synaptopodin knockdown cells detaching from monolayer upon mechanical insult. (J) Western blots showing synaptopodin level unchanged by knockdown of α-actinin-4. (K) Deconvolved optical section showing normal synaptopodin colocalization with E-cadherin in α-actinin-4 knockdown cells (yellow arrowheads). (L) BSA flux assays showing that tension-induced barrier enhancement is absent in α-actinin-4 and synaptopodin knockdown monolayers. (M) BSA flux assays showing decreased ability to withstand mechanical insult in α-actinin-4 and synaptopodin knockdown monolayers. (H, L, and M) Error bars are standard errors. n = 3. (N) Western blots showing E-cadherin, occludin, β-catenin, and p120-catenin levels unchanged in synaptopodin and α-actinin-4 knockdown cells. Bars: (A, D, G, I, and K) 10 µm.