Fig 2: Inhibiting DNA methylation rescues LRIG1 mRNA expression.a Schematic representation of LRIG1 demethylation following ADC (5-Aza-2’-deoxycytidine) treatment. ADC (nucleotide structure, inset) sequesters DNA methyltransferases (DNMTs) resulting in demethylation of LRIG1 DNA and transcriptional activation. b Bar plots showing relative LRIG1 expression levels in ER-negative breast cancer cell lines (BT549, HCC1937, MDA-MB-231) and colon cancer cell line (HCT116) treated with shown concentrations of ADC for 96 h. Expression was determined from the Ct values using the 2-??Ct method after normalisation to GAPDH. LRIG1 fold changes were normalised to 0 µM treatments, which are set to 1.0. c Bar plot showing relative ß-values at LRIG1 Exon 1 and Promoter regions, as assayed by MeDIP-qPCR, in cells treated with vehicle control (VC) or 20 µM ADC for 96 h. Methylation level was determined from the Ct values using the 2-??Ct method after normalisation to 5% input DNA. ß-value was determined by normalisation to HCT116 Exon 1 VC methylation value, which is set to 1.0. Values are mean ± SEM of three (b) or two (c) independent experiments. *P < 0.05 and **P < 0.01. Student’s t test (b, c).

Fig 3: Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 *P = 0.0175, EGF 3 h siCtr vs. sicIAP1 *P = 0.0341; n = 3; two-tailed paired t-test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t-test

Fig 4: Changes in expression of LRIG1 and miR-4295 in GC cells prior to and following DDP treatment. Panel A, miR-4295 expression prior to and following DDP administration detected by RT-qPCR. Panel B, LRIG1 mRNA expression prior to and following DDP administration detected by RT-qPCR. Panel C and D, protein bands and protein expression of LRIG1 prior to and following DDP administration detected by western blot analysis. *P<0.05 vs. expression prior to DDP administration. LRIG1, leucine-rich repeats and immunoglobulin-like domains 1; miR, microRNA; GC, gastric cancer; DDP, cisplatin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± standard deviation of three independent experiments. Two-tailed Student's t-tests were used to analyze the data.

Fig 5: Treatment with SM83 reduces the expression of Snai2 both in vivo and in vitro. a NOD/SCID mice engrafted subcutaneously with 5 × 106 MDA-MB231 were treated with intraperitoneal injection of SM83 (5 mg/kg, 5 times/week) or left untreated (4 mice/group) until the end of the experiment. Schedule of the experiment (upper panel) and tumor volumes (bottom panel) measured twice a week (significant differences in days 24, 27, and 30. *P = 0.0476, 0.0391, and 0.0344, respectively. Unpaired two-tailed t-test). b Six hours after the last injection, mice were killed, nodules collected, and analyzed by western blot to detect the levels of SM83 targets cIAP1, cIAP2, and XIAP. Actin and vinculin are shown as loading controls. c MDA-MB231 cells transfected in vitro with two siRNAs targeting cIAP1 were treated or not with 100 nM SM83 for 1 h. Western blots were performed to evaluate the levels of cIAP1, cIAP2, and XIAP 72 h after transfection. Values show the fold levels of XIAP. d Differentially expressed genes: heat map showing the 50 genes significantly upregulated and the 15 downregulated by SM83 in MDA-MB231 nodules collected as described in Fig. 1a. e Wound-healing experiments performed with MDA-MB231 cells transfected with control (NT1) or Snai2-specific siRNAs (n = 4, **P = 0.0033. Unpaired two-tailed t-test). The graph shows the percentage of gap closure after 24 h of migration. The complete experiment with the other siRNAs tested is shown in Fig. S2a. f The levels of Snai2, downregulated in the GEP shown in Fig. 1d, and LRIG1, upregulated, were evaluated by western blot performed with lysates of MDA-MB231 nodules. Values show the fold levels of Snai2 and LRIG1 normalized to Actin levels. g Levels of Snai2 in MDA-MB231 cells treated with 100 nM SM83 in time-course experiments. Cleavage of p100 NF-kB2 into the p52 form was used to verify the expected activation of the non-canonical NF-kB pathway upon SM83 administration