Pharmacological analysis of the signaling pathway involved in the activation of OR51B4.

<p>Representative calcium imaging traces of HCT116 cells stimulated with Troenan and different specific inhibitors. Grey area represents the duration of the inhibitor applicated. Bar chart showing mean amplitudes of Troenan-induced Ca²⁺ signals in HCT116 cells. <b>(A)</b> Localization of Ca²⁺ by use of EGTA-Ringer. Investigation of different specific inhibitors of calcium signaling upon Troenan stimulation. (<b>B</b>) Gallein (10 μM), <b>(C)</b> U-73522 (10 μM), <b>(D)</b> SQ22.536 (50 μM), <b>(E)</b> H89 (10 μM), <b>(F)</b> RR; Ruthenium red (5 μM), <b>(G)</b> L-cis-diltiazem (150 μM), <b>(H)</b> Mibefradil (10 μM), <b>(I)</b> BTP-2 (25 μM), <b>(J)</b> Thapsigargin (1 μM). N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells. The data are shown as the mean SEM.</p

Transcript abundance of potential effector channels in HCT116 cells determined by RNA-Seq.

<p><b>(A)</b> Bar chart showing the FPKM values of different possible effector channels. Voltage-dependent L- and T-Type channels: CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA2D1, CACNA2D2; CACNA1G, CACNA1H, CACNA1I; CACNB1, CACNB3. Cyclic nucleotide-gated ion channels: CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, CNGB3. Voltage-dependent Ca²⁺ channel: CATSPER1. Transient receptor potential channels: TRPCI, TRPC6, TRPV1, TRPM8, TRPC6, TRPV2, TRPM7, TRPM8. Calcium release-activated calcium channels: ORAI1, ORAI2, ORAI3. <b>(B)</b> Transcript expression of PLC isoforms in the colon cancer cell line HCT116. Bar chart showing FPKM values of different PLCs in the colon cancer cell line HCT116.</p

Expression of OR51B4 in HCT116 cells and colon cancer tissues.

<p><b>(A)</b> Bar chart displays the FPKM values of the most highly expressed ORs in HCT116 cells. <b>(B)</b> Immunocytochemical staining of OR51B4 in HCT116 cells with a specific OR51B4-antibody. Left: HCT116 cells. Right: Negative control: HCT116 cells stained with second antibody alone. Bottom left: Hana3A cells transfected with OR51B4 plasmid. Bottom right: untransfected Hana3A cells. Scale bar: 10 μm. <b>(C)</b> The most highly expressed ORs in NGS analyses, validated by RT-PCR. + = +RT, cDNA;— = -RT, RNA; g = genomic DNA as a control; M = marker. <b>(D)</b> OR51B4 expressed in human colon cancer tissues: RT-PCR analysis shows OR51B4 expression in human colon cancer tissues. Expression in A: Colon tissue B: Colorectal cancer tissue C, D, E: Colon carcinoma tissues. M = marker.</p

Diagram of the proposed signaling cascade that is induced by Troenan stimulation.

<p>PLC: phospholipase C, DAG: diacylglycerol, PKC: protein kinase C, CREB: cAMP response element-binding protein, SOCE: store-operated calcium entry.</p

Impaired actin filament formation and induction of apoptosis upon stimulation of HCT116 cells with Troenan.

<p><b>(A)</b> HCT116 cells treated with Troenan (500 μM) and control cells. <b>(B) and (C)</b> Phalloidin staining of control cells (B) and cells treated with Troenan (300 μM) (C). Scale bar: 10 μm. <b>(D) and (E)</b> Immunocytochemical staining of HCT116 cells with an antibody against caspase-3 after treatment with control (D) or Troenan (300 μM) (E). Cells treated with Troenan (300 μM) for 48 hours. Scale bar: 10 μm. <b>(F)</b> HCT116 cells show decreased serotonin release after application of Troenan (700 μM) for 60 minutes.</p

Western blot analysis of HCT116 cells stimulated with Troenan (300 μM; T) or control (C) for 5 and 25 minutes.

<p><b>(A)</b> Phosphorylation of different isoforms of PKC. <b>(B)</b> Reduced phosphorylation of Stat2, Stat3 and Stat5 upon Troenan stimulation (300 μM). <b>(C)</b> Stimulation of Troenan (300 μM) leads to the time-dependent phosphorylation of p38 MAPK. Reduced phosphorylation was observed for Akt, mTor and Fyn. Troenan stimulation did not affect ERK and SAPK. The total amounts of p38, Akt, ERK, Stat3 and Stat5 and β-actin served as controls. n = 3. <b>(D)</b> Quantification of the mean pixel intensities of the phosphorylated protein kinases Akt, ERK1/2, p38, Stat5 and Stat3. The pixel intensities of duplicates were averaged. Total amounts of the protein kinases were determined and served as controls.</p

Analysis of the Troenan-induced effect in HCT116 cells containing a doxycycline-sensitive OR51B4-knockdown-sequence.

<p><b>(A)</b> Confirmation of knockdown functionality by qRT-PCR and calcium imaging experiments. M = Marker. Stimulation of HCT116/EV (left) and HCT116/10F1 cells (right) with Troenan (100 μM/ 300 μM). <b>(B)</b> Representative calcium signal of HCT116/EV (above) and HCT116/10F1 (below) cells stimulated with Troenan (300 μM) in calcium imaging analysis. <b>(C)</b> Migration analysis via scratch assay with HCT116/EV and HCT116/10F1 cells with and without doxycycline induction. Stimulation of the cells with Troenan (300 μM) for 48 hours. N = 3 assays with 3 dishes. <b>(D)-(G)</b> Proliferation analysis of HCT116/EV (D, E) and HCT116/10F1 (F, G) cells after treatment with Troenan (300 μM) with and without doxycycline induction. Troenan (300 μM) was applied for 72 hours. N = 20.</p

Expression profile of OR51B4 in different carcinoma tissues and–cell lines.

<p><b>(A)</b> Bar chart showing the ranked FPKM values obtained from RNA-Seq data for other tissues and cell lines. <b>(B)</b> Expression of OR51B4 in the transcriptomes of different colon cancer tumors and metastatic tumors.</p

Characterization of Troenan-induced calcium signals in HCT116 cells.

<p><b>(A)</b> Representative image of HCT116 cells stimulated with Troenan (300 μM) in calcium imaging analysis. <b>(B)</b> Number of cells responding to Troenan in different concentrations. <b>(C)</b> Repetitive activation of HCT116 cells upon repetitive Troenan application. <b>(D)</b> Dose-dependent activation of HCT116 cells by Troenan. Troenan was applied at concentrations of 50 μM, 100 μM and 300 μM. Peak amplitudes show the increases in intracellular calcium concentration. To ensure viability of the cells, ATP was applied last, which serves as a positive control. N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells.</p

Physiological effects of Troenan stimulation on HCT116 cells.

<p><b>(A)</b> Analyses of cell migration by scratch assay after Troenan stimulation (300 μM) for 48 hours. <b>(B)</b> Bar chart showing statistical analysis of the area overgrown in scratch assay experiments. n = 3 assays. <b>(C)</b> Monitoring of the cell-index equal to the cell proliferation rate of HCT116 cells incubated with Troenan in different concentrations (50, 100, 150 μM). Dynamic real-time monitoring of cell processes in vivo (xCELLigence RTCA-technology). n = 2 in at least 2 independent experiments.</p