Figure 4

LHFPL3-AS2 downregulation inhibited cell proliferation resulting in G1 cell cycle arrest. (A) mRNA levels (TPM) of CCNE1 and CDK2 in LHFPL3-AS2 knockdown cells (g1 and g2) and controls (gCtl). (B) Western Blot analysis of CCNE1 and CDK2 using GAPDH as a loading control (representative experiment out of 3 replications). Quantifying the protein expression, using ImageJ, and normalizing it to GAPDH indicated a reduction in CCNE1 of 59% with g1 and 24% with g2 and a more modest reduction in CDK2 of 16% with g1 and 18% with g2. (C) Propidium Iodide staining (PI) and FACS analyses of LHFPL3-AS2 knockdown cells (g1 and g2) and controls (gCtl). Percentages of cells in G1, S, and G2/M cell-cycle phases are in a pie chart. One sided t-test, n = 4, *p < 0.05, **p < 0.01. (D) Colony formation assay with representative images (left panel) and quantification (right panel) of the colonies (n = 3). (E) XTT assay absorbance values (OD) (n = 3) show that suppression of LHFPL3-AS2 inhibited Caco-2 cell proliferation. (F) Immunohistochemistry (IHC) staining of Ki-67 in paraffin-embedded sections of Caco-2 3D cysts. Scalebar – 10uM, magnification – × 40.