Figure 9.
Contractomeres and stress fibers are missing in synaptopodin knockdown cells. (A) Immunofluorescence of MDCK wild type (WT) and synaptopodin knockdown (Synpo KD) cells. Apical and basal stress fibers are missing in synaptopodin knockdown cells. Scale bars are 1 μm. (B) Western blots showing reduced cellular levels of myosin IIB and myosin light chain in synaptopodin knockdown cells. Lanes 1 and 3 are whole cell lysate of MDCK parental cells. Lane two is whole cell lysate of synaptopodin knockdown cells. Markers are 150, 100, 75, 25 kD. (C) Scatter plot showing the relationship between synaptopodin and myosin IIB levels in WT (red dots n = 102) and Synpo KD (blue squares n = 119) at the apical junction. Pearson correlation coefficient R is 0.63 between synaptopodin and myosin IIB at WT junction. (D) Myosin IIB is absent from the apical junction in synaptopodin knockdown cells despite the presence of junctional actin. Scale bar is 5 μm. (E) Synaptopodin knockdown (Synpo KD) reduced myosin IIB but not myosin IIA levels at the junction. Measurement of junctional and contractomeric intensities of myosin IIA and IIB immunofluorescence. For upper left and right graph, n (WT) = 102, n (SynpoKD) = 119. For lower left graph, n (WT) = 20, n (SynpoKD) = 32. For lower right graph, n (WT) = 106, n (SynpoKD) = 114. Wilcoxon-Mann-Whitney test P < 0.0001 between WT and Synpo KD for myosin IIB. Wilcoxon-Mann-Whitney test P > 0.1 between WT and Synpo KD for myosin IIA. (F) Contractomeres are missing from junctional vertices (circled) in synaptopodin knockdown cells. Z-stacks are shown in X-Y, Y-Z, and X-Y axes. In synaptopodin knockdown cells, myosin IIB level was lower and α-actinin-4 became cytoplasmic. Scale bars are 5 μm. (G) Myosin IIA and actin remain present at the apical junction in synaptopodin knockdown monolayers. Scale bar is 5 μm. (H) Basal myosin IIA stress fibers are converted into myosin IIA meshworks in synaptopodin knockdown cells. Scale bar is 5 μm.

Contractomeres and stress fibers are missing in synaptopodin knockdown cells. (A) Immunofluorescence of MDCK wild type (WT) and synaptopodin knockdown (Synpo KD) cells. Apical and basal stress fibers are missing in synaptopodin knockdown cells. Scale bars are 1 μm. (B) Western blots showing reduced cellular levels of myosin IIB and myosin light chain in synaptopodin knockdown cells. Lanes 1 and 3 are whole cell lysate of MDCK parental cells. Lane two is whole cell lysate of synaptopodin knockdown cells. Markers are 150, 100, 75, 25 kD. (C) Scatter plot showing the relationship between synaptopodin and myosin IIB levels in WT (red dots n = 102) and Synpo KD (blue squares n = 119) at the apical junction. Pearson correlation coefficient R is 0.63 between synaptopodin and myosin IIB at WT junction. (D) Myosin IIB is absent from the apical junction in synaptopodin knockdown cells despite the presence of junctional actin. Scale bar is 5 μm. (E) Synaptopodin knockdown (Synpo KD) reduced myosin IIB but not myosin IIA levels at the junction. Measurement of junctional and contractomeric intensities of myosin IIA and IIB immunofluorescence. For upper left and right graph, n (WT) = 102, n (SynpoKD) = 119. For lower left graph, n (WT) = 20, n (SynpoKD) = 32. For lower right graph, n (WT) = 106, n (SynpoKD) = 114. Wilcoxon-Mann-Whitney test P < 0.0001 between WT and Synpo KD for myosin IIB. Wilcoxon-Mann-Whitney test P > 0.1 between WT and Synpo KD for myosin IIA. (F) Contractomeres are missing from junctional vertices (circled) in synaptopodin knockdown cells. Z-stacks are shown in X-Y, Y-Z, and X-Y axes. In synaptopodin knockdown cells, myosin IIB level was lower and α-actinin-4 became cytoplasmic. Scale bars are 5 μm. (G) Myosin IIA and actin remain present at the apical junction in synaptopodin knockdown monolayers. Scale bar is 5 μm. (H) Basal myosin IIA stress fibers are converted into myosin IIA meshworks in synaptopodin knockdown cells. Scale bar is 5 μm.

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