# Test file for EGFRSignalingAssay
# Demonstrates per-condition outputs: a vehicle control record that anchors
# the fold-change scale at 1.0 and a cigarette-smoke-extract record, each
# linked to its exposure condition via measured_under.
egfr_signaling_assays:
  - id: "EGFR:001"
    name: "EGFR phosphorylation after smoke exposure"
    has_exposure_condition:
      - id: "EXPOSURE:egfr-vehicle-4h"
        name: "Culture medium vehicle 4 hours (control)"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
      - id: "EXPOSURE:cse-4h"
        name: "Cigarette smoke extract 5% for 4 hours"
        exposure_agent:
          id: "ENVO:01000838"
          name: "smoke"
        exposure_concentration:
          value: "5"
          unit:
            id: "UO:0000187"
            name: "percent"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
    has_specified_output:
      - id: "EGFR:001-output-control"
        name: "Vehicle control EGFR signaling measurements"
        experimental_group: vehicle_control
        measured_under: "EXPOSURE:egfr-vehicle-4h"
        egfr_phosphorylation_y1068:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        total_egfr_protein:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        egfr_phosphorylation_y1173:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        downstream_kinase_activation: "ERK1/2 and AKT at basal activity"
        erk_phosphorylation:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        akt_phosphorylation:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        egfr_ligand_expression:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        egfr_membrane_localization:
          value: "45"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 4
          variability:
            variability_type: standard_error
            value: "3.8"
            unit:
              id: "UO:0000187"
              name: "percent"
      - id: "EGFR:001-output-cse"
        name: "Cigarette smoke extract EGFR signaling measurements"
        experimental_group: treated
        measured_under: "EXPOSURE:cse-4h"
        egfr_phosphorylation_y1068:
          value: "3.2"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
          variability:
            variability_type: standard_error
            value: "0.4"
            unit:
              id: "STATO:0000169"
              name: "fold change"
        total_egfr_protein:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        egfr_phosphorylation_y1173:
          value: "2.1"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        downstream_kinase_activation: "ERK1/2 and AKT activated"
        erk_phosphorylation:
          value: "2.8"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        akt_phosphorylation:
          value: "1.5"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        egfr_ligand_expression:
          value: "1.9"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 4
        egfr_membrane_localization:
          value: "78"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 4
          variability:
            variability_type: standard_error
            value: "4.1"
            unit:
              id: "UO:0000187"
              name: "percent"
    normalization_reference: "beta-actin"
    assay_date: "2024-05-08"
    informs_on_key_event:
      id: "KE:ke1-egfr-activation"
      name: "EGFR activation"
      biological_action: activated
      level_of_biological_organization: molecular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-005"
      name: "Primary HBE ALI culture"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:wb-001"
        name: "Western blot for phospho-EGFR"
        detection_method: "chemiluminescence"
        antibodies_used:
          - "anti-pEGFR Y1068 (Cell Signaling #3777)"
          - "anti-total EGFR (Cell Signaling #4267)"
        normalization_method: "total protein normalization"

# Analysis layer: EGFR membrane localization is reported as a percentage, so
# the change relative to control is an absolute difference in percentage
# points rather than a fold change.
response_comparisons:
  - id: "COMP:EGFR001-y1068"
    name: "Change in EGFR Y1068 phosphorylation, CSE vs vehicle"
    derived_from_assay: "EGFR:001"
    compared_measurement: "egfr_phosphorylation_y1068"
    control_output: "EGFR:001-output-control"
    treated_output: "EGFR:001-output-cse"
    change_type: fold_change
    change_direction: increased
    change_value:
      value: "3.2"
      unit:
        id: "STATO:0000169"
        name: "fold change"
    p_value: 0.002
    statistical_test: "two-sample t-test"
    derivation: reported_in_paper
  - id: "COMP:EGFR001-membrane-localization"
    name: "Change in EGFR membrane localization, CSE vs vehicle"
    derived_from_assay: "EGFR:001"
    compared_measurement: "egfr_membrane_localization"
    control_output: "EGFR:001-output-control"
    treated_output: "EGFR:001-output-cse"
    change_type: absolute_difference
    change_direction: increased
    change_value:
      value: "33"
      unit:
        id: "UO:0000187"
        name: "percent"
    p_value: 0.005
    statistical_test: "two-sample t-test"
    derivation: computed