Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A-B. CCK-8 and EdU assays were used to detect proliferation in HEI-OC1 cells treated with LPS. As detected by CCK-8 and EdU assays, cell proliferation was markedly suppressed by LPS treatment compared to the control group. Scale bar = 100 µm in B. C. Apoptosis in HEI-OC1 cells treated with LPS was assessed via flow cytometry analysis. The apoptotic rate was increased significantly by LPS treatment. D. Western blot analysis of BCL-2 and BAX expressions in HEI-OC1 cells treated with LPS. The BAX protein level increased while the BCL-2 protein level decreased after LPS treatment. E. Western blot analysis of HDAC2 protein expression in HEI-OC1 cells treated with LPS, DEX, or DEX+TA. HDAC2 protein levels were decreased in HEI-OC1 cells treated with LPS alone, while its levels were partly reversed by DEX treatment and totally reversed by treatment with both DEX and TA (**P < 0.01).

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: CCK-8 Assay, Control, Flow Cytometry, Western Blot, Expressing

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A. q-PCR and western blot analyses of NRF2 and HDAC2 expressions in HEI-OC1 cells treated with LPS, DEX or DEX+TA. TA increased NRF2 and HDAC2 mRNA and protein levels in DEX-treated HEI-OC1 cells (**P < 0.01). B. RT-qPCR was used to check gene over-expression of NRF2 in HEI-OC1 cells. NRF2 was successfully over-expressed in HEI-OC1 cells. C. HDAC2 mRNA level was detected via RT-qPCR in HEI-OC1 cells over-expressed NRF2. We detected an increased HDAC2 mRNA level in HEI-OC1 cells over-expressed NRF2 (**P < 0.01). D. Potential NRF2-binding site in the sequence of HDAC2 promoter was predicted by JASPAR database. Based on JASPAR database ( https://jaspar.genereg.net/ ) analysis, we obtained putative NRF2-binding sites in the sequence of HDAC2 promoter. The NRF2-binding site with the highest prediction score is located at 419–429 bases (ATGACACTCCA) in HDAC2 promoter sequence. E. ChIP detected enrichment of HDAC2 promoter in immunoprecipitate with Anti-NRF2. ChIP assay presented an abundant enrichment of HDAC2 promoter in immunoprecipitates with Anti-NRF2, verifying the binding between NRF2 and HDAC2 promoter. F. Luciferase activity of different reporter constructs in NRF2-overexpressing 293T cells was detected. G. DNA pull-down assay followed by western blot was used to detect NRF2 expression in the complex pulled down by Bio-HDAC2 promoter-WT. NRF2 over-expression increased the luciferase activity of HDAC2 promoter-WT while not changing the HDAC2 promoter-MUT activity (mutated sequence: TACTGTGAGGT at 419–429 bases). **P < 0.01.

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: Western Blot, Quantitative RT-PCR, Over Expression, Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Pull Down Assay, Expressing

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A. RT-qPCR and western blot were used to measure FOXP3 expression in LPS-treated HEI-OC1 cells. FOXP3 mRNA and protein levels were increased in response to additional TA treatment (P < 0.01). B. Gene over-expression efficiency of FOXP3 in HEI-OC1 cells was tested via RT-qPCR. FOXP3 was over-expressed in HEI-OC1 cells. C. GR expression in FOXP3-over-expressing HEI-OC1 cells was measured via western blot. GR protein level was also increased in FOXP3-over-expressing HEI-OC1 cells. D. NRF2 expression in FOXP3-over-expressing HEI-OC1 cells was measured via RT-qPCR. Elevated NRF2 mRNA level was observed in FOXP3-over-expressed HEI-OC1 cells. E. Potential FOXP3-binding sites in NRF2 promoter sequence were predicted by JASPAR database. According to JASPAR database analysis, the predicted FOXP3-binding site with the highest prediction score is located at 864–870 bases (GCAAACA) in NRF2 promoter sequence. F. Luciferase activity of different reporter constructs in FOXP3-over-expressing 293T cells was detected by luciferase reporter assay. The luciferase activity of pGL3-NRF2 promoter-WT was enhanced by up-regulation of FOXP3 in 293T cells, while that of pGL3-NRF2 promoter-MUT (mutated sequence: CGTTTGT at 864–870 bases) was not affected. G. ChIP assay was performed to detect the physical binding between FOXP3 and NRF2 promoter. ChIP assay uncovered that NRF2 promoter was highly enriched in Anti-FOXP3-precipitated complex. H. DNA pull-down assay followed by western blot was used to detect FOXP3 expression in the complex pulled down by Bio-NRF2 promoter-WT. DNA pull-down assay validated the effectiveness of the binding sites between FOXP3 and NRF2 promoter. I. The knock-down efficiency of si-NRF2 plasmids was checked via RT-qPCR. All three siRNAs had high efficacy of NRF2 knock-down. J. HDAC2 expression was measured via RT-qPCR and western blot in HEI-OC1 cells. The increased mRNA and protein levels of HDAC2 caused by FOXP3 over-expression were reversed by depletion of NRF2. K. FOXP3 expression was knocked down in DEX-treated HEI OC1 cells. The mRNA level of NRF2 was measured in DEX-treated HEI OC1 cells after FOXP3 knock-down by RT-qPCR. L. Protein levels of FOXP3 and NRF2 were measured in DEX-treated HEI OC1 cells after FOXP3 knock-down. The mRNA and protein levels of NRF2 increased by TA treatment while the levels were re-decreased by silencing of FOXP3 (**P < 0.01).

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Reporter Assay, Pull Down Assay, Knockdown

Journal: bioRxiv

Article Title: Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoid in lipopolysaccharide-treated HEI-OC1 cells through modulation of Foxp3/Nrf2 signaling pathway

doi: 10.1101/2024.08.19.608552

Figure Lengend Snippet: A. Interference efficiency of si-HDAC2 plasmids in LPS-induced HEI-OC1 cells was checked via RT-qPCR and western blot. si-HDAC2-1 plasmid was used in the subsequent experiments for its highest interference efficiency. B-C. CCK-8 and EdU assays were performed to assess cell proliferation. Scale bar: 100 µm. HDAC2 knock-down abrogated the promoting effect of FOXP3 over-expression on cell proliferation. D-E. Western blot of BAX and BCL-2 expressions and flow cytometry analysis were performed to evaluate apoptosis. Elevated BCL-2 protein levels and declined BAX protein levels resulting from FOXP3 up-regulation were recovered by HDAC2 deficiency. Moreover, the apoptosis rate reduced by FOXP3 over-expression was restored by HDAC2 knock-down (**P < 0.01).

Article Snippet: Biotinylated wild-type or mutated HDAC2 promoter/NRF2 promoter probes (Bio-HDAC2 promoter-WT/MUT or Bio-NRF2 promoter-WT/MUT) or NC probes (Bio-NC) synthesized by Ribobio were added to 1% Triton X-100, followed by heating at 95 °C for 2 min and cooling in an ice bath for 3 min.

Techniques: Quantitative RT-PCR, Western Blot, Plasmid Preparation, CCK-8 Assay, Knockdown, Over Expression, Flow Cytometry