The Hippo pathway drives the cellular response to hydrostatic pressure (2022)
Figure 2

Figure 2. YAP/TAZ determine cell size and mediate cellular response to hydrostatic pressure.

A Cell volume of WT and Y/T DKO clones at steady state measured using DHM. Each dot represents a single cell. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. Data from three independent experiments. ***p<0.001, p=0.0917 (Y/T DKO #1 vs #2).

B Comparison of cell volume of WT and LATS1/2 DKO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. **p=0.0018 (WT vs LATS1/2 DKO #1), ***p<0.001 (WT vs LATS1/2 DKO #2), p=0.0557 (LATS1/2 DKO #1 vs #2).

C Comparison of cell volume of WT and MST1/2 DKO at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.2731.

D HEK293A NF2 KO cells response to 100mbar cyclic hydrostatic pressure. Each dot represents a single cell and Error bars represent mean ± 95% CI. Data pooled from three independent experiments. Mann-Whitney U test. p=0.4072.

E Comparison of cell volume of WT and TEAD KO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. p>0.9999 for all comparisons.

F Maxima and minima are identified to calculate average percentage change in cell volume in response to hydrostatic pressure using measurements obtained by DHM.

G Representative WT and Y/T DKO single cell volume change in response to hydrostatic pressure recorded by DHM.

H Average change in cell volume in response to hydrostatic pressure in WT and Y/T DKO clones. Each dot represents a single cell. Images and data were acquired at the onset of oscillating pressure. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post hoc. ***p < 0.001 (for both comparisons), p=0.1450 (Y/T DKO #1 vs #2).

I Representative images of Flipper-TR labelled HEK293A WT (top), Y/T DKO #1 (middle) and Y/T DKO #2 (bottom) are shown obtained using fluorescence lifetime imaging microscopy (FLIM) (ex. 485, em. 600)

J Average fluorescence lifetime of Flipper-TR probe quantified using fluorescence lifetime imaging microscope (FLIM) (ex. 485, em. 600). HEK293A wildtype and Y/T DKO clones #1 and #2 were labelled with 2μM Flipper-TR for 15 minutes prior to imaging. The average lifetime measurements were obtained by fitting the photon histogram with a double exponential and the longest lifetime was extracted to report plasma membrane tension, as previously described (Colom et al., 2018). Each dot represents fluorescence lifetime of a single cell and data shown is pooled from three independent experiments (mean±95% CI). Kruskal-Wallis test with Dunn's post hoc. **p=0.0027 (WT vs Y/T DKO#1), *p=0.0136 (WT vs Y/T DKO #2), p>0.9999 (Y/T DKO#1 vs #2).

K Representative immunofluorescence images showing MYC-tagged YAP expression in WT YAP re-expressing cells and S94A mutant YAP re-expressing cells compared to Y/T DKO vector control. 95% were positive for expression of YAP or YAP mutant. Scale bar = 20µm.

L Western blot showing total YAP, TAZ and CYR61 levels in WT YAP and S94A mutant YAP re-expressing cells compared to WT and Y/T DKO vector control.

M Average change in cell volume in response to hydrostatic pressure in WT YAP and S94A mutant YAP re-expressing cells relative to Y/T DKO. Each dot represents a single cell. Error bars represent mean ± 95% CI. Data pooled from three independent experiments. Kruskal-Wallis test with Dunn's post hoc. p<0.001 (vector vs. WT YAP), p=0.4912 (vector vs. S94A YAP), p=0.0327

N Average change in cell volume in response to 100mbar cyclic hydrostatic pressure in WT and TEAD KO clones. Each dot represents a single cell. Data pooled from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post hoc. ***p < 0.001 (for both comparisons), p>0.9999 (TEAD KO #1 vs #2).

O WT and Y/T DKO cell volume were measured at steady-state and the average change in cell volume was quantified using the DHM as in previous experiments, these are labelled (-). Cells were then subjected to cyclic hydrostatic pressure for 2 hours and their cell volume was quantified to determine whether prior exposure to hydrostatic pressure ('pre-conditioning') would change their cell volume response, these are labelled (+). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal-Wallis with Dunn's post hoc. ns= p>0.9999.

P The average percentage change in cell volume was quantified under the same experimental conditions as in "O". The average change in cell volume in response to cyclic hydrostatic pressure of cells with no previous exposure to hydrostatic pressure (labelled as (-)) was quantified using DHM and compared to those with prior exposure to hydrostatic pressure (labelled (+)). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal-Wallis with Dunn's post hoc. **p=0.006 (WT), p=0.4173 (Y/T DKO), ns= p>0.9999.