Figure 5

LHFPL3-AS2 knockdown results in abnormal multipolar mitotic spindle formation. (A) Fluorescence staining using anti-tubulin (red) and nuclear Hoechst (blue) of LHFPL3-AS2 CRISPRi knockdown and gCtl Caco-2 cysts. White arrows indicate mitotic spindles. Scalebar—20uM, magnification—x60oil. (B) Schematic representation of a normal bipolar mitotic spindle. (C) Caco-2 grown as 3D cysts were treated with 20 nM Taxol for 24 h to arrest cells during mitosis. Left panel—representative images of the mitotic spindles in LHFPL3-AS2 knockdowns and controls (gCtl). White arrows indicate centrosomes. Right panel—Percentage of cells with bipolar spindles, multipolar spindles, or monopolar spindles captured during mitosis. Fisher exact test between the fraction of cells with bi-polar spindles vs. multi/mono-polar spindles. ****P ≤ 0.0001. (D) Schematic cartoon summarizing LHFPL3-AS2's potential role in regulating epithelial polarity, proliferation, and mitotic spindle formation. Reduction of LHFPL3-AS2 affects apicobasal positioning of cytoskeleton protein (actin) and tight/adherens junction proteins. Loss of cellular polarity and abnormal cellular positioning likely underlies the abnormal mitotic spindle and centrosome arrangement, resulting in G1 cell cycle arrest, reduced cellular proliferation, and the robust subsequent inhibition of transcription and translation of many key epithelial genes including CTNNB1 and TCF4.