Figure 2 | Scientific Reports

Figure 2

From: Reduced LHFPL3-AS2 lncRNA expression is linked to altered epithelial polarity and proliferation, and to ileal ulceration in Crohn disease

Figure 2

LHFPL3-AS2 knockdown results in robust loss of Caco-2 luminal cyst formation. (A) LHFPL3-AS2 knockdown was achieved using CRISPRi and 2 gRNAs, and those cells were compared with gCtl. qPCR confirmed a reduction in LHFPL3-AS2, after normalization to GAPDH. The two-sided t-test is shown. ****P < 0.0001. (B) Light microscopy imaging (× 10) of LHFPL3-AS2 knockdown or Caco-2 and gCtl cells after seeding them as a monolayer on a culture plate for 3 days, showing spontaneous rounding and clumping in the knockdown cells. Scalebar—100uM, magnification – × 10. (C) F-actin Phalloidin staining (green) and nuclear staining (blue) of LHFPL3-AS2 knockdown or gCtl cells after seeding them as a monolayer on a culture plate. The upper panel shows the xy-axis views and the lower panel shows a 3D reconstruction of z-stack images. LHFPL3-AS2 knockdown cells show a rounding appearance as demonstrated on the z-axis when grown as a monolayer, with less confluency and more clumping. Magnification x63oil, scalebar-20 µM. (D) Schematic representation and bright field imaging of LHFPL3-AS2 knockdown or Caco-2 and gCtl 3-dimensional (3D) cyst grown in Matrigel for 5 days, with lumen/apical part inside. Scalebar—20uM, magnification – × 40. (E) Haematoxylin and eosin (H&E) staining of Caco-2 cysts shows that most LHFPL3-AS2 knockdown cysts have no lumen. Scalebar—20uM, magnification – × 40. (F) Quantification of (D) indicating the percentage of cysts with a single central lumen, no lumen, or multiple lumens were quantified. Fisher exact test was performed comparing the number of cysts with a single lumen to those with no lumen or with multiple small lumens. (G) Phalloidin staining for F-actin (green) and nuclear Hoechst (blue) staining of Matrigel-grown cysts. Magnification x63oil, scalebar-20uM. (H) Line plots denoting the intensity of actin staining along the apical/basal axis of indicated cyst along the white line, from the apical to basolateral side. The image shows a representative experiment out of 6 replications (shown in Supplementary Fig. S3).

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