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. 2023 Jan 7;122(18):3630–3645. doi: 10.1016/j.bpj.2023.01.005

Figure 2.

Figure 2

Indirect methods of Rho activation cause acinar eversion. (A) Confocal immunofluorescent images of 9-day-old acini untreated with doxycycline (-Dox) or a 7-day-old acinus treated with doxycycline for the indicated time (+Dox) to induce expression of mCherry-KASH1. Middle: an acinus in the process of everting. Bottom: a fully everted acinus. Nuclei were stained with blue. Scale bars, 20 μm. (B) Bar graph of the percentage occurrence of podocalyxin localization in untreated (-Dox) or treated (+Dox, 48 h, and +Dox, 72 h) acini, separately scored based on the presence of podocalyxin on the interior surface in acini with a single lumen, podocalyxin on the interior surface in acini multiple lumens, and podocalyxin on the exterior surface in acini with no lumens. At least 70 acini from three independent experiments were scored for each condition. Error bars represent ±SEM (∗p < 0.05; one-way ANOVA test with post hoc Tukey (HSD) test). (C) Confocal fluorescent images of a 10-day-old GFP-podocalyxin-expressing acinus treated with 2 μg/mL doxycycline to induce mCherry-KASH1 expression at T = 0 h (see Videos S7, S8, and S9), or (D) confocal fluorescent images of a 10-day-old GFP-podocalyxin-expressing acinus treated with 2 μg/mL doxycycline to induce mCherry-KASH1ΔPPPL expression (which does not disrupt the LINC complex); images are shown at the indicated times after this treatment (see Video S10). The results in (C) represent eight acini out of the 20 assayed acini from a total of three independent experiments (lumens in the 12 acini did not collapse over the time frame of the experiment). (E) Western blot shows total and active RhoA in absence and presence of doxycycline to induce mCherry KASH1 expression in MDCK cells in culture. The cells in 2D culture were treated with 100 μg/mL of doxycycline until a monolayer was formed, followed by the RhoA pull-down assay. (F) Confocal fluorescent images of GFP-podocalyxin and stained for β1-integrin of a 9-day-old untreated acinus and a 7-day acinus treated with 40 μM Y-27632 for 48 h. DNA was stained with Hoechst 33342 (blue). Scale bar, 20 μm. (G) Comparison of acinar frequency with podocalyxin in the apical surface corresponding to the different conditions in (F). At least 70 acini from three independent experiments were scored for each condition. Error bars represent ±SEM (∗p < 0.05; one-way ANOVA test with post hoc Tukey (HSD) test).