Fig 2: NRF2 activation desensitises RTK signaling pathway to HER2 inhibitors Pertuzumab and Trastuzumab. (a) Immunoblot analysis showing tBHQ dependent recovery of RTK signaling following its inhibition by HER2 inhibitors. Exponentially growing cells were either left untreated (UT) or treated with combination of HER2 inhibitors, Pertuzumab and Trastuzumab at concentration of 20 μg/mL (RTKi), or with cotreatment of 200 μM tBHQ (RTKi + T) for 4 h before and processed for immunoblotting using relevant antibodies (Table 1). Ponceau stain of the same blot was used as loading control. (b) Bar chart showing total HER2, phospho-HER2, total HER3, phospho-AKT, phospho-ERK, phospho-NRF2, and BID levels in PEO1 and SKOV3 cell lines by quantifying immunoblot signal intensities obtained in (a) and normalised to the value of UT and expressed as fold change. Blue bars show ratio of phospho-HER2 to HER2 (upper panels) and cleaved BID to intact BID (lower panels).

Fig 3: HER3 copy number increased in TR cells, and interfering with HER3 expression restored sensitivity to tarloxotinib-E. (A) HER3 expression levels are higher in TR cells than in parental H1781 cells, regardless of tarloxotinib-E exposure. (B) The HER3 DNA copy number of TR cells was approximately 1.5 times higher than that of parental H1781 cells. (C) TR cells were resensitized to tarloxotinib-E by combined treatment with siHER3. (D) The phosphorylation of HER3 and its downstream signal, p-Akt, were suppressed by the combination of tarloxotinib-E and siHER3. On the other hand, the decrease in p-Erk1/2 was limited. tar-E, tarloxotinib-E; TR, tarloxotinib-E-resistant; siHER3, small interfering HER3; p-Erk1/2, phosphor-Erk1/2; t-Erk1/2, total-Erk1/2; p-Akt, phosphor Akt; t-Akt, total-Akt.

Fig 4: HER receptors are activated by MEK inhibitor in MAP3K1; MAP2K4 wild-type cells. a Control and MAP3K1 or MAP2K4 knockout H358 or HCT116 cells were treated with 2 μM selumetinib for 72 h and lysates were western blotted for p-HER3, HER3, p-JUN, JUN, MAP2K4 or MAP3K1, p-ERK, ERK. HSP90 served as a control. b Control and MAP3K1 or MAP2K4 knockout MDA-MB-468 cells were treated with 0.02 μM trametinib for 72 h. The levels of p-HER3, HER3, p-JUN, JUN, p-ERK, ERK, MAP3K1 and MAP2K4 were determined by western blot analysis. c Two breast cancer cell lines of indicated MAP2K4 mutation status were treated with 2 μM selumetinib for 72 h and lysates were subjected to phospho-RTK activation analysis. Dots labeled 1-4 represent duplicate blots of p-EGFR, p-HER2, p-HER3 and p-HER4, respectively. d MDA-MB-468 cells were treated with selumetinib for 72 h, then RNA was extracted and qRT-PCR analysis was performed for HER receptor transcripts. e Two individual shRNAs targeting JUN were introduced into MDA-MB-468 cells by lentiviral transduction. Ctrl and JUN knockdown cells were treated with 2 μM selumetinib for 72 h. The levels of phospho-HER1-4 and HER1-4 receptors, p-JUN, JUN, p-ERK and ERK were determined by western blot analysis. HSP90 served as a loading control. f MDA-MB-468 cells were cultured for two weeks in medium containing increasing concentration of selumetinib alone, dacomitinib (8 nM) alone, or combination of selumetinib and dacomitinib. After this, cells were fixed and stained

Fig 5: ERBB2 controls cervical cancer cell viability and invasion by regulating PIK3CA protein expression. (A) Schematic diagram of the ERBB2-ERRB3 complex interacting with PI3K(p85), thereby promoting the downstream phosphorylation of AKT and mTOR. (B) IP in cervical cancer cell lysates with antibodies against ERBB3 or IgG control. Expression levels of ERBB3, ERBB2 and PI3K(p85) in the IP fraction were assessed via WB. PIK3CA mRNA expression in transfected (C) HeLa and (D) SiHa cells assessed via RT-qPCR. GAPDH was used as the housekeeping control. PIK3CA, p-AKT/AKT and p-mTOR/mTOR protein expression in transfected (E) HeLa and (F) SiHa cells assessed via WB. GAPDH was used as the loading control. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit-8. (I) Sphere-formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. siCtrl or Ctrl vec; †P<0.05 and ††P<0.01 vs. siERBB2 or ERBB2 vec. Data were analyzed via one-way ANOVA. IP, immunoprecipitation; WB, western blotting; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; vec, vector; ERBB2, Erb-B2 Receptor Tyrosine Kinase 2; p-, phosphorylated; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α.