p3xflag p62 var2  (Addgene inc)

Journal: Advanced Science

Article Title: Targeting Immunoproteasome in Polarized Macrophages Ameliorates Experimental Emphysema Via Activating NRF1/2‐P62 Axis and Suppressing IRF4 Transcription

doi: 10.1002/advs.202405318

Figure Lengend Snippet: ONX‐0914 Suppresses M1 Polarization via NRF1 and NRF2‐P62 Signaling Pathway. A) Differential binding of transcription factors between M1+ONX‐0914 and M1 predicted by TOBIAS. This plot indicates a significant increase in NRF1 (MAFG::Nfe2l1) and NRF2 (MAF::Nfe2l2) binding score following ONX‐0914 treatment. B) Mouse genome NRF1 and NRF2 motifs obtained from JASPAR 2022 database. C) Gene Ontology enrichment analysis of genes upregulated by ONX‐0914 treatment in M1 phenotype of PMs. The y‐axis represents the GO terms, and the x‐axis indicates the gene count. D) Heatmaps displaying proteasome‐related genes, the NRF1 downstream regulated genes, in M0, M0+ONX‐0914, M1, and M1+ONX‐0914 group (n = 4). E) Volcano plot showing DEGs between ONX‐0914‐treated and untreated M1 AMs. Genes meeting dual thresholds of FDR <0.05 and log 2 (fold change) >1 are highlighted in red, indicating significant upregulation or downregulation after ONX‐0914 treatment. F) Protein expression level of NRF1 analyzed by Western blotting in ONX‐0914‐treated M1 AMs. Data was obtained from 3 independent samples in each group. G) Heatmaps displaying anti‐oxidative stress‐related genes, the NRF2 downstream regulated genes, in M0, M0+ONX‐0914, M1, and M1+ONX‐0914 group of PMs (n = 4). H) Representative IF images of NRF2 after applying 0.2 µM ONX‐0914 to AMs for 24 h. I) After AMs were pretreated with 0.2 µM ONX‐0914 for 6 h, and then stimulated with 250 µg mL −1 dosage of CSE for 24 h, the gene Nfe2l2 was examined by RT‐qPCR (n = 3). J and K) Transcriptional changes in genes associated with NRF1‐increased binding sites (J) and NRF2‐increased binding sites (K) after ONX‐0914 treatment in M1 of AMs. The plot demonstrates that the regulatory activity of upregulated genes significantly surpasses that of downregulated genes (n = 3). L) After silencing the expression of NRF1 with siRNA and treating M1‐polarized AMs with 0.2 µM ONX‐0914, the expression of Nfe2l1, Sqstm1 , and M1 marker genes ( Nos2, Il12b ) were detected by RT‐qPCR (n = 3). M) After silencing the expression of NRF2 with siRNA and treating M1‐polarized AMs with 0.2 µM ONX‐0914, the expression of Nfe2l2, Sqstm1 , and M1 marker genes ( Nos2, Il12b, Il1b, Tnf ) were detected by RT‐qPCR (n = 3). N) Integrative Genomics Viewer (IGV) plot illustrates the binding landscape of the NRF2 transcription factor to Sqstm1 in M0, M0+ONX‐0914, M1 and M1+ONX‐0914. The upper 4 tracks display the signal intensity of NRF2 binding to Sqstm1 , and the lower 4 tracks display the specific locations where NRF2 binding peaks were identified, which suggest the potential regulatory roles in Sqstm1 gene expression after ONX‐0914 treatment in M1 macrophages. O) CUT&Tag‐qPCR shows that the binding affinity of NRF2 to Sqstm1 enhancer and promoter is significantly increased under ONX‐0914 treatment in M1 macrophages. P) RT‐qPCR analysis detected Nfe2l2 , Sqstm1 and M1 marker genes Nos2 , Il12b , Il1b , and Il6 in each group (n = 3). After silencing the expression of NRF2 with siRNA and overexpression of P62 with p3xFLAG‐P62 var2 plasmid, 0.2 µM ONX‐0914 was used to treat M1‐polarized AMs, and the expression of Nfe2l2 , Sqstm1 , and M1 marker genes ( Nos2 , Il12b , Il1b and Il6 ) were detected by RT‐qPCR (n = 3). Q) After silencing the expression of NRF2 with siRNA and using 0.2 µM ONX‐0914 pretreated CSE (250 µg mL −1 ) stimulation of AMs, Sqstm1 and M1 marker genes ( Nos2, Il12b, Il1b, Tnf ) were detected by RT‐qPCR (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The plasmids used in this study included p3xFLAG‐P62 var2 (#204576, Addgene) and pMIG‐IRF4 (#58987, Addgene).

Techniques: Binding Assay, Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Marker, Gene Expression, Over Expression, Plasmid Preparation