Fig 2: Septins recruit ß-catenin to E-cadherin. (A) Caco-2 cells were transfected with control shRNA, shSEPT2, shSEPT6, and shSEPT7, respectively. Cells were harvested and subjected to immunoprecipitation with E-cadherin antibody or IgG, and the immunoprecipitates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS?PAGE) followed by western blotting using indicated antibodies. Note that E-cadherin brought down less ß-catenin from SEPT2/6/7-depleted cells than control cells. (B) Quantitative analysis of the relative ß-catenin intensity in shSEPT2/6/7 groups compared to control group. Data represent mean ± SEM from three independent experiments. Statistical significance was tested by two-sided t-test. ***P < 0.001, ****P < 0.0001. (C) Coomassie Brilliant Blue staining of SDS?PAGE gel was used to assess the quality and quantities of the purified recombinant peptides TAT-GFP-His, TAT-GFP-SEPT2-M-His (TAT-GFP-2M-His; amino acids 35?306), TAT-GFP-SEPT6-M-His (TAT-GFP-6M-His; amino acids 39?305), and TAT-GFP-SEPT7-M-His (TAT-GFP-7M-His; amino acids 31?296). (D) HEK293T cells expressing FLAG-SEPT2/6/7 and GFP-ß-catenin were subjected to immunoprecipitation with FLAG antibody in the presence of TAT-GFP or TAT-GFP-SEPT2/6/7-M (TAT-GFP-2M/6M/7M) for overnight incubation and immunoblotted with His, FLAG, and GFP antibodies, respectively. Immunoblotting analysis showed that the interaction of ß-catenin and SEPT2/6/7 was perturbed by the addition of TAT-GFP-SEPT2/6/7-M (TAT-GFP-2M/6M/7M). (E and F) Caco-2 monolayers were cultured to 70% confluence followed by introducing 3 µM TAT-GFP or TAT-GFP-SEPT2/6/7-M (TAT-G-2M/6M/7M) into DMEM at 37°C for overnight. Cells were fixed after 100% confluence followed by immunostaining with indicated antibodies. Note that the staining of ß-catenin/E-cadherin is diminished upon SEPT2/6/7-M peptide competition.

Fig 3: Septins are necessary for efficient epithelial cell polarity/barrier formation. (A) Schematic presentation of the method for TEER test of Caco-2 monolayers with drug treatment. Caco-2 single cells were plated onto transwell and grown for several days until confluent monolayers were established. After monolayers achieved maximal TEER, cells were treated with different drugs and TEER was monitored every 2 h. (B) Monolayers on transwell were treated with DMSO, FCF, Nocodazole, or Lat.B and TEER was monitored every 2 h. Data represent mean ± SEM from three independent experiments. (C) Schematic presentation of the method for TEER test of Caco-2 monolayers expressing control shRNA, shSEPT2, shSEPT6, or shSEPT7 under Ca2+ switch assay. Monolayers were cultured in Ca2+-free minimum essential medium (MEM) with 10% dialyzed FBS for 16 h to break the Ca2+-dependent cell junction and minimal TEER was achieved. Afterwards, the culture medium was changed to DMEM containing 10% fetal bovine serum (FBS) to reconstruct cell junction and TEER was monitored every 2 h. (D) Development of TEER after Ca2+ switch in control, SEPT2/6/7-depleted, and E-cadherin-depleted cells. Data represent mean ± SEM from three independent experiments. (E) Working model accounting for the function of septin cytoskeleton in epithelial apical?basal polarization and adherens junction integrity maintenance. SEPT2/6/7 filaments bind with E-cadherin and ß-catenin directly to promote the stability of E-cadherin-dependent adherens junction at lateral membrane.

Fig 4: SEPT6 regulated prostate cancer cell behavior through UBC. (a) CCK-8 was used to detect cell proliferation. (b) Clonal formation assay detected cell clone number. (c) Cell cycle at 24 h was detected by flow cytometry. (d) WB was performed to detect proliferation-related protein PCNA and Ki67 expressions. (e) The expression levels of cell cycle proteins CDK1, CCNA2, MCM10, and E2F1 were detected by qRT-PCR. (f) qRT-PCR detected proliferation-related protein HIST1H1A, HIST1H3B, BRCA1, and AURKB expressions. *P < 0.05 vs. the NC group; #P < 0.05 vs. the oe-SEPT6 group.

Fig 5: Septin filaments localize at adherens junction in polarized epithelial cells. (A) Schematic illustration of 2D cell culture. Caco-2 single cells were plated onto coverslip and grown for 3 days to form 100% confluent monolayers, which have distinct cell junction. (B) Schematic presentation of the organization of SEPT2/6/7 hetero-hexamer. (C and D) Representative immunofluorescence images of Caco-2 monolayers. Cells were fixed, permeabilized, and stained with anti-SEPT2/6/7 (red) and anti-ß-catenin or anti-E-cadherin (green), respectively. Note that SEPT2/6/7 and adherens junction markers colocalize at cell junction. A single section through the middle of monolayers is shown. Scale bar, 20 µm. (E) Schematic presentation of 3D cell culture. Caco-2 single cells were seeded in Matrigel and grown for 6 days to allow cyst formation with an open lumen. (F and G) Representative immunofluorescence images of Caco-2 3D cysts. Cysts were fixed and stained with anti-SEPT2/6/7 (red) and anti-ß-catenin or anti-E-cadherin (green). Note that SEPT2/6/7 and adherens junction markers colocalize at lateral membrane in 3D cyst model. A single confocal section through the middle of cysts was projected. Scale bar, 20 µm.