Additional file 4: Figure S3. of A machine learning classifier trained on cancer transcriptomes detects NF1 inactivation signal in glioblastoma

Training Distribution Matching (TDM) transformation of RNAseq results of The Cancer Genome Atlas Glioblastoma parameter sweep for stochastic gradient descent logistic classifier with elastic net penalty. (A) Training and testing area under the receiver operating characteristic curve (AUROC) is given for each parameter tested. All accuracies are presented following 5-fold cross validation after 100 random initializations. (B) The l1 mixing parameter with the optimal alpha and (C) the classifier performance across all random starts for the best hyperparameters. (PNG 724 kb

Additional file 5: Figure S4. of A machine learning classifier trained on cancer transcriptomes detects NF1 inactivation signal in glioblastoma

Cohen’s D effect size estimates across five fold cross validation parameters for all 100 iterations of the TDM transformed ensemble classifier. The effect size for the test set is consistently lower than the training set (left). Additionally, the training and testing decision functions for gold standard NF1 deficient vs. NF1 wildtype samples shows a difference in mean estimates (right). The decision function represents the raw score of all samples as applied to the respective classifiers through each of the 100 iterations of five fold cross validation on the TCGA training set. (PNG 580 kb

Additional file 6: Table S2. of A machine learning classifier trained on cancer transcriptomes detects NF1 inactivation signal in glioblastoma

Ranked genes contributing greater than two standard deviations to ensemble classifier. (XLSX 27 kb

Titration of GFP-eIF2α BacMam.

<p>(A) U-2 OS cells were mixed with different amount of GFP-eIF2α BacMam and plated to a 384 well culture plate for overnight. Cells were then treated with either DMSO or 2 μM Tg for 1 h at 37°C 5% CO₂. LanthaScreen was performed to detect eIF2α phosphorylation. Data was the average of 4 repeats. (B) U-2 OS cells were transduced with GFP-eIF2α BacMam in a culture flask with the optimized MOT for overnight. After trypsinized and washed, cells were resuspended to different cell density and plated into a 384 well plate. After the treatment with Tg for 2.5 h at 37°C and 5% CO₂, LanthaScreen assay was performed to detect the phosphorylation of GFP-eIF2α. Data was the average of 3 repeats.</p

LanthaScreen assay signal was PERK dependent.

<p>U-2 OS cells were transduced with GFP-eIF2α BacMam under the optimized conditions for overnight. Cells were then trypsinized and plated into a 384 well plate. Different doses of a specific PERK inhibitor GSK2606414 (A) or an inactive analog (B) was incubated with cells for 30 min at 37°C and 5% CO₂. DMSO, 1 μM Tg or 5 μM Tn was then added and incubated for 2 h at 37°C 5% CO₂. Phosphorylation of GFP-eIF2α was analyzed with LanthaScreen assay. Data was the average of 2 repeats.</p

Compounds have insignificant activity in IRE1 branch.

<p>HT1080 cells pretransduced with XBP-1 splicing reporter gene were treated with compounds at different concentrations for 3 h at 37°C and 5% CO₂. Luciferase activity was measured with Steady Glo reagents after the treatment. Data were average of 8 repeats.</p

Compounds increase Heme Oxygenase-1 (HO-1) expression in a NRF2 dependent pattern.

<p>(A) NHBE cells were treated with 10 μM of compound for 1 h or 3 μM of compound for 24 h at 37°C and 5% CO₂, respectively. Tg (0.5 μM) and hemin (3 μM) served as controls. HO-1 protein was then determined. (B) NHBE cells were transfected with 25 nM non-target siRNA, PERK siRNA or NRF2 siRNA for 48 h, then treated with compounds at 3 μM for 24 h. HO-1 mRNA level was then analyzed. (C) NHBE cells transfected with non-targeting, NRF2 or PERK siRNA for 48 h, treated with DMSO, 0.5 μM Tg, 3 μM compound B, C or hemin for 24 h. HO-1 protein level was determined. Data was representative of 3 experiments.</p

Robustness features of the LanthaScreen assay.

<p>U-2 OS cells were transduced with GFP-eIF2α BacMam for overnight in flasks. Cells were then trypsinized, washed and resuspended for plating into 384 well plates for compound treatment. The low control was DMSO only, the high control was 1 μM Tg. All the conditions were the optimized final conditions as described in results.</p><p>Robustness features of the LanthaScreen assay.</p

Compounds increase NQO-1 protein expression in a NRF2 dependent pattern.

<p>(A) NHBE cells were treated with 10 μM compound for 1 h or 3 μM compound for 24 h at 37°C and 5% CO₂. NQO-1 protein level was determined by ELISA. Stars indicate p value ≤0.001 (n = 3). (B) NHBE cells were transfected 25nM siRNA for 48 h, treated with DMSO, 0.5μTg, 3 μM compound B or C for 24 h. NQO-1 mRNA level was then determined by Real-Time PCR and was normalized with DMSO control (n = 3). (C) NHBEs were transfected 25 nM siRNA for 48 h, treated with DMSO, 0.5 μM Tg, 3 μM compound B or C for 24 h. NQO-1 protein level was determined by Western blot. Data was representative of 3 experiments.</p

Systemic IL-6 and MCP-1 are elevated in patients with large burn injuries and organ dysfunction.

<p>(A) Scatter plot analysis of systemic IL-6 levels between mild (<15% TBSA, Black circles) and moderate/severe (≥15% TBSA, Red squares) patients at 0–48 HPA. (B) Scatter plot analysis of systemic MCP-1 levels between mild (<15% TBSA, Black circles) and moderate/severe (≥15% TBSA, Red squares) patients at 0–48 HPA. (C) Scatter plot analysis of systemic IL-6 levels between burn patients that suffer pulmonary distress(0–24 HPA, <357 SpO₂/FiO₂ ratio) and patients that did not (>357 SpO₂/FiO₂ ratio). (D) Scatter plot analysis of systemic MCP-1 levels between burn patients that suffer pulmonary distress(0–24 HPA, <357 SpO₂/FiO₂ ratio) and patients that did not (>357 SpO₂/FiO₂ ratio). (E) Line plot representing longitudinal analysis of systemic IL-6 accumulation from 0–48 HPA and 72–144 HPA timepoints. (F) Line plot representing longitudinal analysis of systemic MCP-1 accumulation from 0–48 HPA and 72–144 HPA timepoints. Error bars represent ± SEM. T-tests were performed to determine statistical significance for (A-D). **** represents <i>P</i> < .0001, *** represent <i>P</i> < .001, * represent <i>P</i> < .05.</p