Journal: Oncogene
Article Title: Preclinical validation of Alpha-Enolase (ENO1) as a novel immunometabolic target in multiple myeloma
doi: 10.1038/s41388-020-1172-0
Figure Lengend Snippet: a Oligonucleotide array analysis: MM cells were cocultured with pDCs for 48 h, separated using anti-CD138 antibody and flow sorting, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Data processing: the CEL files were obtained using Affymetrix Microarrays Suite 5.0 software. GeneChip 5.0 (Affymetrix, Santa Clara, CA) was utilized to scan, quantify, and analyze the scanned image. GeneChip software automatically calculated intensity values for each probe cell, and marked a presence or absence call for each mRNA. Algorithms in the software used probe cell intensities to calculate an average intensity for each set of probe pairs representing a gene, which correlates with the amount of mRNA. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared, and a heat map was generated (>1.5-fold change in transcript is considered significant, CI > 95%). The expression profile of ENO1 transcript is shown. b Quantification of ENO1 expression: normalized ENO1 gene enrichment in pDC-MM cell co-culture versus MM cells alone is presented [1.772-fold upregulation; n = 3; CI > 95%]. c Validation and Quantification of ENO1 gene expression by RT-qPCR: MM cells were cocultured with pDCs for 48 h; separated using anti-CD138 antibody and flow sorting, and harvested. Poly RNA was subjected to RT-qPCR using Luna Universal 1-step RT-qPCR kit (New England BioLabs, MA, USA) following manufacturer protocol using an Applied Bioscience 7500 Fast Real-Time PCR System (Thermo Scientific Inc). ENO1 gene expression was quantified from the raw data using ΔΔCT and utilizing GAPDH as the housekeeping reference gene control. The bar graph denotes ENO1 gene expression in MM cells cultured in the presence vs absence of pDCs (mean ± SD; p < 0.05). Inset: Analysis of qPCR amplicons (130 bp) on a 2.5% agarose gel stained with GelRed Nucleic Acid Gel Stain (Biotium Inc, USA). Lane-1: 1 kb DNA ladder; Lanes-2 and –3: ENO1 expression in MM cultured in the absence and presence of pDCs, respectively. d Expression data from different stages of plasma cell neoplasm collected using Affymetrix Human Genome U133A [HG-U133A] Array platform based on NORMAL (N = 4), MGUS (N = 11), and multiple myeloma (MM: N = 73) samples. The data is presented as fold change (FC) in each group versus normal. e Survival analysis of TT2 MM patient cohorts based on high and low expression of ENO1 gene. The gene expression analysis is based on TT2 patients survival data collected using [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. The survival difference between high vs low expression groups is significant for the duration of the study (p = 0.012). [Note D-E: The analysis of data was based on the information available on the following website: http://www.canevolve.org/AnalysisResults/AnalysisResults.html].
Article Snippet: GeneChip 5.0 (Affymetrix, Santa Clara, CA) was utilized to scan, quantify, and analyze the scanned image.
Techniques: Microarray, Software, Expressing, Cell Culture, Generated, Co-Culture Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining
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![a Oligonucleotide array analysis: MM cells were cocultured with pDCs for 48 h, separated using anti-CD138 antibody and flow sorting, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Data processing: the CEL files were obtained using Affymetrix Microarrays Suite 5.0 software. GeneChip 5.0 (Affymetrix, Santa Clara, CA) was utilized to scan, quantify, and analyze the scanned image. GeneChip software automatically calculated intensity values for each probe cell, and marked a presence or absence call for each mRNA. Algorithms in the software used probe cell intensities to calculate an average intensity for each set of probe pairs representing a gene, which correlates with the amount of mRNA. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared, and a heat map was generated (>1.5-fold change in transcript is considered significant, CI > 95%). The expression profile of ENO1 transcript is shown. b Quantification of ENO1 expression: normalized ENO1 gene enrichment in pDC-MM cell co-culture versus MM cells alone is presented [1.772-fold upregulation; n = 3; CI > 95%]. c Validation and Quantification of ENO1 gene expression by RT-qPCR: MM cells were cocultured with pDCs for 48 h; separated using anti-CD138 antibody and flow sorting, and harvested. Poly RNA was subjected to RT-qPCR using Luna Universal 1-step RT-qPCR kit (New England BioLabs, MA, USA) following manufacturer protocol using an Applied Bioscience 7500 Fast Real-Time PCR System (Thermo Scientific Inc). ENO1 gene expression was quantified from the raw data using ΔΔCT and utilizing GAPDH as the housekeeping reference gene control. The bar graph denotes ENO1 gene expression in MM cells cultured in the presence vs absence of pDCs (mean ± SD; p < 0.05). Inset: Analysis of qPCR amplicons (130 bp) on a 2.5% agarose gel stained with GelRed Nucleic Acid Gel Stain (Biotium Inc, USA). Lane-1: 1 kb DNA ladder; Lanes-2 and –3: ENO1 expression in MM cultured in the absence and presence of pDCs, respectively. d Expression data from different stages of plasma cell neoplasm collected using Affymetrix Human Genome U133A [HG-U133A] Array platform based on NORMAL (N = 4), MGUS (N = 11), and multiple myeloma (MM: N = 73) samples. The data is presented as fold change (FC) in each group versus normal. e Survival analysis of TT2 MM patient cohorts based on high and low expression of ENO1 gene. The gene expression analysis is based on TT2 patients survival data collected using [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. The survival difference between high vs low expression groups is significant for the duration of the study (p = 0.012). [Note D-E: The analysis of data was based on the information available on the following website: http://www.canevolve.org/AnalysisResults/AnalysisResults.html].](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2141/pmc07132141/pmc07132141__nihms-1574464-f0001.jpg)