Presentation on theme: "KM12C shRNA SCRAMBLE KM12C shRNA CDH17 Negative control E-cadherin"— Presentation transcript:
1KM12C shRNA SCRAMBLEKM12C shRNA CDH17Negative controlE-cadherinCell numberCell numberMean fluorescence intensity104101102103Mean fluorescence intensity104101102103KM12SM shRNA SCRAMBLEKM12SM shRNA CDH17Cell numberCell numberMean fluorescence intensity104101102103Mean fluorescence intensity104101102103Figure S1. KM12 cells do not express E-cadherin before or after CDH17 silencing. KM12C and SM cells expressing shRNAs scrambled or targeting CDH17 were subjected to flow cytometry assays using anti-E-cadherin antibody and Alexa 488 labeled-secondary antibodies. Fluorescence was analyzed in a Coulter Epics XL cytofluorometer. No surface expression was detected.
2ABInvasive cellsCell migration speed (μm/h)KM12CKM12SMKM12CKM12SMKM12CKM12SMKM12CKM12SMshRNA SCRAMBLEshRNA CDH17 #60shRNA SCRAMBLEshRNA CDH17 #60Figure S2. Silencing of CDH17 does not modify KM12 cell migration and invasion. A) KM12 cell lines were cultured to confluence in Matrigel-coated plates (0.4 μm/mm²), and a 1 mm-wide wound was done across the monolayer. Migration speed of the cells was calculated from the distance covered after scratching in two days, as quantified in the pictures taken immediately or 48h after the scratching, divided by 48 (number of hours) and by 2 (because cells advanced in two fronts) . B) Invasion across Matrigel of the indicated KM12 cell lines towards serum. After 48 h of incubation, non-invading cells were removed and invasive cells were fixed, stained and counted under a microscope. In both experiments, data represent the mean + SD of 2 independent experiments.
3ControlCDH17ControlCDH17ControlCDH17siRNA:CDH17RhoGDICaco2SW480SW620Figure S3. Assessment of silencing of CDH17 in Caco2, SW480 and SW620 cells. These cell lines were transfected with siRNAs against CDH17, or with control siRNAs. After 48h, cells were lysed, extracts were resolved by SDS-PAGE gels and subjected to Western blot analysis to detect CDH17. RhoGDI was used as loading control.
4Mean fluorescence intensity 1041011021036.97.14.412.518.104.22.168Cell numberKM12C shRNA SCRAMBLEKM12C shRNA CDH17 #60KM12SM shRNA CDH17 #60KM12SM shRNA SCRAMBLEMean fluorescence intensityNegative Controlα2-integrinβ1-integrinFigure S4. Silencing of CDH17 does not alter α2β1 integrin expression on KM12 cell surface. KM12 cell lines were detached with 2 mM EDTA in PBS and incubated with antibodies against α2 or β1 integrin subunits or with control antibodies. After washing, cells were incubated with secondary antibodies labeled with FITC. Cells were analyzed in a cell fluorometer to assess cell surface expression of afore mentioned integrin subunits.
5RhoGDIβ-cateninpY229-p120 cateninKM12C shRNA SCRAMBLEKM12C shRNA CDH17KM12SM shRNA SCRAMBLEKM12SM shRNA CDH17p120 cateninFigure S5. Expression of β-catenin and p120-catenin is not altered after CDH17-silencing. KM12 cell lines were lysed, protein extracts were resolved by SDS-PAGE and analyzed by western blot to assess expression of β-catenin and phosphorylated p120-catenin. After stripping, membrane was reincubated with antibodies against total p120-catenin. Control loading was done with anti-RhoGDI antibodies.