FSP1 Promoter Assay in M1-Polarized MDMs

FSP1 promoter assay was done as described in ref. ^{28 (link)}. FSP1 promoter construct plasmid containing 1.0–1.3 kb insert, corresponding to the 5′-flanking sequence located ~1.3 kb upstream and up to 200 bp downstream of the transcription initiation site of human FSP1 (NM_002961) HPRM17407-PG04 (GeneCopoeia) inserted upstream of the GLuc reporter gene was used. M1-polarized human MDMs polarized were co-transfected with the FSP1 promoter reporter construct and miR-21 mimic using Lipofectamine LTX (Thermo Fisher Scientific). Media were collected 48 h post transfection and the secreted GLuc and SEAP activities were measured with the Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia) according to the manufacturer’s protocol using the Berthold Luminometer (Berthold Technologies). Normalized promoter activity has been presented as the ratio of GLuc to SEAP activities.

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Sinha M., Sen C.K., Singh K., Das A., Ghatak S., Rhea B., Blackstone B., Powell H.M., Khanna S, & Roy S. (2018). Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue. Nature Communications, 9, 936.

Publication 2018

Lipofectamine LTX Secrete-Pair Dual Luminescence Assay Kit Berthold Luminometer

Assay Human Lipofectamine Luminescence assay Mdms Plasmid Reporter gene Transcription initiation site Transfection

Corresponding Organization :

Other organizations : The Ohio State University Wexner Medical Center, Lung Institute, The Ohio State University

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Variable analysis

independent variables

MiR-21 mimic

dependent variables

Normalized promoter activity (ratio of GLuc to SEAP activities)

control variables

FSP1 promoter construct plasmid containing 1.0–1.3 kb insert, corresponding to the 5′-flanking sequence located ~1.3 kb upstream and up to 200 bp downstream of the transcription initiation site of human FSP1 (NM_002961) HPRM17407-PG04 (GeneCopoeia)
M1-polarized human MDMs

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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