Ab50887

The cell lysates were collected, and 30 mg protein of each sample was separated using 10% SDS-PAGE. The protein was then electrotransferred onto Polyvinylidene Fluoride (PVDF) membrane (Millipore, Boston, MA, USA). The PVDF membrane was sealed in 10% skimmed milk. Then the membrane was incubated with primary antibodies, including anti-TNC (Abcam, ab108930, 1:1000 dilution), anti-GAPDH (Abcam, ab181602, 1:10,000 dilution), and anti-ERK (Abcam, ab184699, 1:1000 dilution)), anti-p-ERK (Abcam, ab201015, 1:1000 dilution), anti-MMP9 (Abcam, ab76003, 1:1000 dilution), anti-MMP2 (Abcam, ab92536, 1:1000 dilution), anti-E-cadherin (Abcam, ab1416, 1:1000 dilution), anti-N-cadherin (Abcam, ab18203, 1:1000 dilution), anti-vimentin (Abcam, ab92547, 1:1000 dilution), anti-snail (Abcam, ab216347, 1:1000 dilution), anti-clumping (Abcam, ab27568, 1:1000 dilution) and anti-TWIST1 (Abcam, ab50887, 1:1000 dilution) overnight. Then the membrane and horseradish peroxidase-conjugated secondary antibody (Abcam AB6721, 1:10,000 dilution) were incubated for 2 h at room temperature. Chemiluminescence detection reagent (Millipore, Boston, MA, USA) was used to visualize protein bands.

Patients with early‐stage breast cancer were identified from the London Breast Cancer Database on the basis of having either DCIS only (stage 0) or DCIS with an associated invasive component (stage I; ≤ 2 cm, and either pN0 or pN0mi). Cohort 1 consisted of 186 patients with low‐, intermediate‐ or high‐grade DCIS with no invasion or early invasion (stage 0 or stage I). Cohort 2 consisted of 118 patients with non‐high‐grade DCIS with the aforementioned characteristics, with either no invasion or early invasion (stage 0 or stage I). Clinicopathological variables for cohort 1 and cohort 2 patients entered into this study are listed in Table 1. There was no overlap between patients in cohort 1 and cohort 2. The study was conducted under a protocol approved by the Western University Health Sciences Research Ethics Board and the Clinical Research Impact Committee of Lawson Health Research Institute. Immunohistochemical staining was conducted using the following antibodies: TBX3 (Abcam, Cambridge, MA, USA; ab99302; 1/200), SLUG (Abcam; ab27568; 1/750), and TWIST1 (Abcam; ab50887; 1/750). Details relating to immunohistochemistry protocol and quantification methods are located in the supplementary material, Supplementary materials and methods.

Western blot was performed to explore the effects of transfections on the expression of Twist1 protein. At 24 h post-transfection, SHP-77 cells were harvested and total proteins were extracted from 10⁵ cells using RIPA solution (GenePharma) and protein concentrations were measured using bicinchoninic acid assay (BCA) assay (GenePharma). All samples were incubated with boiling water for 12 min to denature proteins, following by electrophoresis (10% SDS-PAGE gel) to separate proteins. PVDF membranes were used to perform protein transfer and blocking was achieved by incubating the membranes with 5% non-fat milk for 2 h at room temperature. Following that, membranes were first incubated with anti-Twist1 (1: 2000, ab50887, Abcam) anti-GLUT1 (1: 2000, ab15309, Abcam) rabbit primary antibodies for 15 h at 4 °C, followed by incubation with goat HRP (IgG) (1:2000; ab6721; Abcam) secondary antibody for 2 h at 25 °C. ECL Western Blotting Substrate Kit (ab65623, Abcam) was used to develop signals and grey values were processed using Image J v1.46 software.