Schematic representation of the 2∶1 DPPS:PI5P lipid preparation protocol.

<p>Schematic representation of the 2∶1 DPPS:PI5P lipid preparation protocol.</p

Confirmation of inhibitors.

<p>(A) <i>i</i>, The IC₅₀ inhibition curves of the tyrphostin analogues identified from the Lopac library are shown. Numbers refer to the tyrphostin analog and IC₅₀s were determined to be as follows: tyrphostin I-OMe-AG-538 (2 µM), tyrphostin 51 (5 µM), tyrphostin AG 112 (13 µM), tyrphostin AG 538 (14 µM), tyrphostin AG 808 (18 µM), tyrphostin 47 (20 µM), tyrphostin AG 537 (32 µM), tyrphostin 23 (45 µM), tyrphostin AG 555 (50 µM), tyrphostin AG 698 (50 µM), tyrphostin AG 490 (89 µM), tyrphostin AG 494 (89 µM), tyrphostin AG 1478 (100 µM). <i>ii</i>, Structures of the four most potent tyrphostin analogs. (B) The IC₅₀ curves with standard deviation error bars (N = 2) of tyrphostin I-OMe-AG-538 in the PI5P4Kα assay (squares) and the counterscreen (open circles).</p

ATP competition with tyrphostin I-OMe-AG-538.

<p>The IC₅₀ of tyrphostin I-OMe-AG-538 for PI5P4Kα is plotted against the [ATP]/K_m. The K_m of ATP is 5 µM, and seven concentrations were evaluated.</p

Lipid dependence, overnight stability and control compound.

<p>(A) The overnight (16 hour) stability of the assay reagents at 4°C when the enzyme and lipid were premixed, stored separately or made up fresh as compared to a no enzyme and 5 µM ADP (representing 0% and 100% conversion, respectively). The error bars represent the standard deviation (N = 2). (B) The PI5P lipid dependence of the PI5P4Kα enzyme reaction. The error bars represent the standard deviation (N = 2) and are not discernable on the plot. (C) and (D) Tyrphostin AG-82 (AG82) was identified as a weak inhibitor of PI5P4Kα (decreases the enzyme activity by 75%) by a radiometric assay that uses γ-³²P-ATP and PI5P and measures the radiolabeled enzymatic product, PI(4,5)P₂ after the separation by thin layer chromatography. Five additional compounds were tested and found not to significantly inhibit PI5P4Kα (AG17 =  tyrphostin AG-17, AG18 =  tyrphostin AG-18, MP = mycophenolate, PVB = purvalanol B and SU6668). All compounds were tested at 100 µM, except for PVB, which was tested at 10 µM due to solubility limitations at higher concentrations. The raw image and the extracted data are shown in (C) and (D), respectively. The commercial PI5P substrate predominantly contains two palmitate groups with a very small amount of deacylated lipid lyso-PI5P that contains only one palmitate group. The intense top spots in (C) represent the PI(4,5)P₂ product with two palmitate groups, and the faint spots below represent the product with just one palmitate group.</p

Schematic representation of the PI5P4K reaction using PI5P as the substrate.

<p>The additional carrier substrate DPPS is not shown.</p

Additional file 3: Figure S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Distributions of molecular alterations according to histology in Cohort 1. Figure S2. Kaplan-Meier analysis for Group A cases stratified by 1p/19q status. Figure S3. Kaplan-Meier analyses for GBM cases in Cohorts 1 and 2. (PPTX 172 kb

Additional file 2: Table S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Molecular and clinical characteristics of Cohort 1 (n = 758). Table S2. Molecular and clinical characteristics of GBM cohort (n = 453). Table S3. Univariate and multivariate Cox regression analyses for Group A (IDH mutated-TERT mutated) tumors in Cohort 1 (n = 155). Table S4. Univariate and multivariate Cox regression analyses for Group B (IDH mutated-TERT wild-type) tumors in Cohort 1 (n = 131). Table S5. Univariate and multivariate Cox regression analyses for Group C (IDH wild-type-TERT wild-type) tumors in Cohort 1 (n = 237). Table S6. Univariate and multivariate Cox regression analyses for Group D (IDH wild-type-TERT mutated) tumors in Cohort 1 (n = 235). Table S7. Univariate and multivariate Cox regression analyses for GBM in Cohort 1 (n = 260). Table S8. Univariate and multivariate Cox regression analyses for GBM in Cohort 2 (n = 193). Table S9. Background of combined GBM cohort stratified by TERT and MGMT status (n = 453). Table S10. Survival time and WHO grade in each molecular subgroup of Cohort 1 (n = 758). (XLSX 254 kb

Additional file 1: of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Supplementary Information. (DOCX 141 kb