SNX27 is a negative regulator of membrane associated E-cadherin expression in CRC cells.
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(A) Whole cell lysate immunoblot analysis siSNX27 knockdown in SW480 cells. Cells were transfected using identical siRNA oligo sequences that were used in the screen for SNX27 or ZEB1 (SMARTpool) and protein levels were assessed 72 hours later. The blot was probed with antibodies against E-cadherin, SNX27 and β-tubulin (loading control) and is representative of 4 individual experiments. (B and C) Quantification of SNX27 (B) and E-cadherin protein (C) levels upon siSNX27 knockdown in SW480 cells (n = 4). Protein levels were determined using densitometry against the loading control β-tubulin and displayed as the mean ± SEM For SNX27 (B) ***p<0.001 for all samples vs mock control; for E-cadherin (C) ***p<0.001 for SNX27 si #1; **p<0.01 (p = 0.006 for SNX27si #2, and p = 0.005796 for ZEB1 si), *p<0.05 (p = 0.02 and p = 0.028 for SNX27si #3 and 4, respectively), for all samples vs mock control, paired one-tailed Student’s t-test. (D and E) SNX27 depletion promotes junctional E-cadherin in SW480 cells (D) and HCT116 cells (E). E-cadherin (Ecad) (green), SNX27 (red) and nuclei (DAPI) (blue). Scale bar 50μm. (F and G) SNX27 regulates cell adhesion through an interaction in the SNX27PDZ domain. SNX27-eGFP expression disrupts junctional E-cadherin in SW480+APC cells (F) but PDZ-domain mutant, SNX27-H114A-eGFP expression does not (G). Cell contacts are indicated by arrows. Junctional staining is absent in SNX27-eGFP expressing cells ** (F) but are intact in SNX27-H114A-eGFP expressing cells # (G). SNX27-eGFP and SNX27-H114A-eGFP (green), E-cadherin (red), nuclei (DAPI) (blue). Scale bar 50μm, left hand panels and 80μm, enlarged inset, right hand panels. (H) Whole cell lysate immunoblot analysis of SNX27 and E-cadherin levels in Colo320, SW480 and SW480+APC cells. β-tubulin serves as a loading control. (I) Immunoblot analysis of ZEB1, E-cadherin and β-tubulin in SW480 and SW480+APC cells. Shown are cropped blots, representative of three independent experiments. (J) ZEB1 expression upon siSNX27 knockdown in SW480 cells. Immunoblot analysis: the blot was probed with E-cadherin, ZEB1, SNX27 and β-tubulin (loading control) antibodies and ZEB1 protein levels quantified (mean ± SEM, n = 3; unpaired one-tailed Student’s t-test, *p<0.05, p values are indicated). (K) ZEB1 does not regulate SNX27 levels. SW480 cells were transfected with siZEB1 (SMARTpool) and protein levels were assessed 72 hours later. The blot was probed with SNX27 and β-tubulin (loading control) antibodies and are representative of two independent experiments. Quantitation is shown below (mean ± SEM, n = 2). Shown are cropped blots. Uncropped blots are included in S1 Raw images.