#METABOLOMICS WORKBENCH DATATRACK_ID:4082 STUDY_ID:ST000275 ANALYSIS_ID:AN000439 VERSION 1 CREATED_ON 08-09-2023 #PROJECT PR:PROJECT_TITLE Metabolic analysis of Parp1 ko/wt Saline & Bleo Mouse Lung Fibroblasts and Human PR:PROJECT_TITLE IPF & Normal Lung Fibroblasts PR:PROJECT_TYPE Glycolysis/TCA/Nucleotide analysis (tissue/cells) PR:PROJECT_SUMMARY Hedgehog signaling plays important roles in cell development and PR:PROJECT_SUMMARY differentiation. In this study, the ability of Sonic Hedgehog (SHH) to induce PR:PROJECT_SUMMARY myofibroblast differentiation was analyzed in isolated human lung fibroblasts, PR:PROJECT_SUMMARY and its in vivo significance was evaluated in rodent bleomycin-induced pulmonary PR:PROJECT_SUMMARY fibrosis. The results showed that SHH could induce myofibroblast differentiation PR:PROJECT_SUMMARY in human lung fibroblasts in a Smo- and Gli1-dependent manner. Gel shift PR:PROJECT_SUMMARY analysis, chromatin immunoprecipitation assay, and site-directed mutagenesis PR:PROJECT_SUMMARY revealed that a Gli1 binding consensus in the ?-SMA gene promoter was important PR:PROJECT_SUMMARY for mediating SHH-induced myofibroblast differentiation. Analysis of Hedgehog PR:PROJECT_SUMMARY reemergence in vivo revealed that of all three Hedgehog isoforms, only SHH was PR:PROJECT_SUMMARY significantly induced in bleomycin-injured lung along with Gli1. The induction PR:PROJECT_SUMMARY of SHH was only noted in epithelial cells, and its expression was undetectable PR:PROJECT_SUMMARY in lung fibroblasts or macrophages. Transforming growth factor (TGF)-? induced PR:PROJECT_SUMMARY SHH significantly in cultured alveolar epithelial cells, whereas SHH induced PR:PROJECT_SUMMARY TGF-? in lung fibroblasts. Pulmonary fibrosis and ?-smooth muscle actin (?-SMA) PR:PROJECT_SUMMARY expression were significantly reduced in mice that were Smo deficient only in PR:PROJECT_SUMMARY type I collagen–expressing cells. Thus, the reemergence of SHH in epithelial PR:PROJECT_SUMMARY cells could result in induction of myofibroblast differentiation in a PR:PROJECT_SUMMARY Smo-dependent manner and subsequent Gli1 activation of the ?-SMA promoter. PR:INSTITUTE University of Michigan PR:DEPARTMENT Deaprtment of Pathology PR:LABORATORY Sem H. Phan PR:LAST_NAME Hu PR:FIRST_NAME Biao PR:ADDRESS Ann Arbor, MI PR:EMAIL biaohu@med.umich.edu PR:PHONE 734-7635731 PR:DOI http://dx.doi.org/10.21228/M8359Z #STUDY ST:STUDY_TITLE Metabolic analysis of Parp1 ko/wt Saline & Bleo Mouse Lung Fiboblasts and Human ST:STUDY_TITLE IPF & Normal Lung Fiboblasts (Part 2) ST:STUDY_TYPE Glycolysis/TCA/Nucleotide analysis. Ceramide analysis for parp1 wild type lung ST:STUDY_TYPE tissue/Cells after saline or bleomycin treatment. ST:STUDY_SUMMARY This study is a part of series performed for the same researcher through ST:STUDY_SUMMARY pilot/feasibility grant program, so the publication is relevant reference for ST:STUDY_SUMMARY other studies (ST000143, ST000183) This specific experiment is a small pilot ST:STUDY_SUMMARY study to establish method performance, it includes four biological replicas of ST:STUDY_SUMMARY identical cell cultures after the identical treatment and a single tissue ST:STUDY_SUMMARY sample. ST:INSTITUTE University of Michigan ST:DEPARTMENT Deaprtment of Pathology ST:LABORATORY Sem H. Phan ST:LAST_NAME Hu ST:FIRST_NAME Biao ST:ADDRESS Ann Arbor, MI ST:EMAIL biaohu@med.umich.edu ST:PHONE 734-7635731 ST:SUBMIT_DATE 2014-06-11 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:SPECIES_GROUP Mammal #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - S00014994 Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes SUBJECT_SAMPLE_FACTORS - S00014995 Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes SUBJECT_SAMPLE_FACTORS - S00014996 Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes SUBJECT_SAMPLE_FACTORS - S00014997 Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes SUBJECT_SAMPLE_FACTORS - S00014998 Sample type:Tissue | Genetic background:B6 wt | bleomycin:yes #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE Cells #TREATMENT #SAMPLEPREP SP:SAMPLEPREP_SUMMARY - SP:SAMPLEPREP_PROTOCOL_FILENAME Gly-TCA-nucleotides_analysis_protocol-2015-03-09.docx #CHROMATOGRAPHY CH:METHODS_ID AQM020 CH:METHODS_FILENAME QTOF-002-HILIC-35min-1mm.m CH:INSTRUMENT_NAME Agilent 1260 CH:COLUMN_NAME Phenomenex Luna NH2 (150 x 1mm,3um) CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:LABORATORY_NAME MRC2 (University of Michigan) AN:ANALYSIS_TYPE MS AN:ACQUISITION_ID AQM011 AN:ACQUISITION_PARAMETERS_FILE ALPHA_KETO_ACIDS-FULL.M AN:PROCESSING_PARAMETERS_FILE EX00310-MassHunterQuant-GlyTCA-DataAnalysis-GCMS-Method.m #MS MS:INSTRUMENT_NAME Agilent 6530 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM (500ul of extraction solvent) MS_METABOLITE_DATA_START Samples S00014994 S00014995 S00014996 S00014997 S00014998 Factors Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes Sample type:Cell culture | Genetic background:B6 wt | bleomycin:yes Sample type:Tissue | Genetic background:B6 wt | bleomycin:yes Alanine 6.2436 5.8979 6.0826 5.8632 4.4470 alpha-Ketoglutarate 0.3880 0.3836 0.4108 0.4049 0.2233 Aspartate 7.7196 7.1164 8.4866 7.9883 17.3353 fumarate 0.5219 0.5178 0.5209 0.5249 0.5905 Glutamate 8.6397 7.9679 8.8075 8.5750 4.3956 glutamine 12.1172 10.6305 12.8258 14.2882 3.0844 Lactate 30.9503 39.2068 32.3482 43.0763 10.2946 Oxaloacetate pyruvate 2.0451 2.3407 2.0106 2.4476 1.1894 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant Alanine 5950 C00041 ALA UM_Target_ID alpha-Ketoglutarate 51 C00026 AKG UM_Target_ID Aspartate 5960 C00049 ASP UM_Target_ID fumarate 444972 C00122 FUM UM_Target_ID Glutamate 611 GLU UM_Target_ID glutamine 5961 C00064 GLN UM_Target_ID Lactate 107689 C00186 LAC UM_Target_ID Oxaloacetate 970 C00036 OAA UM_Target_ID pyruvate 1060 C00022 PYR UM_Target_ID METABOLITES_END #END