Additional file 1 of Patients’ and physicians’ awareness of clinical symptoms and disease severity in tuberous sclerosis complex

Additional file 1. Flow chart of included questionnaires and Supplementary Tables

DataSheet1_Analyzing the postulated inhibitory effect of Manumycin A on farnesyltransferase.docx

Manumycin A is postulated to be a specific inhibitor against the farnesyltransferase (FTase) since this effect has been shown in 1993 for yeast FTase. Since then, plenty of studies investigated Manumycin A in human cells as well as in model organisms like Caenorhabditis elegans. Some studies pointed to additional targets and pathways involved in Manumycin A effects like apoptosis. Therefore, these studies created doubt whether the main mechanism of action of Manumycin A is FTase inhibition. For some of these alternative targets half maximal inhibitory concentrations (IC50) of Manumycin A are available, but not for human and C. elegans FTase. So, we aimed to 1) characterize missing C. elegans FTase kinetics, 2) elucidate the IC50 and Ki values of Manumycin A on purified human and C. elegans FTase 3) investigate Manumycin A dependent expression of FTase and apoptosis genes in C. elegans. C. elegans FTase has its temperature optimum at 40°C with KM of 1.3 µM (farnesylpyrophosphate) and 1.7 µM (protein derivate). Whilst other targets are inhibitable by Manumycin A at the nanomolar level, we found that Manumycin A inhibits cell-free FTase in micromolar concentrations (Ki human 4.15 μM; KiC. elegans 3.16 μM). Furthermore, our gene expression results correlate with other studies indicating that thioredoxin reductase 1 is the main target of Manumycin A. According to our results, the ability of Manumycin A to inhibit the FTase at the micromolar level is rather neglectable for its cellular effects, so we postulate that the classification as a specific FTase inhibitor is no longer valid.</p

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

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

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

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

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