Journal: Frontiers in Molecular Biosciences

Article Title: SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice

doi: 10.3389/fmolb.2021.725319

Figure Lengend Snippet: Six1 is upregulated in the proximal tubular epithelial cells (TECs) after unilateral ischemia/reperfusion injury (IRI). (A) Serum creatinine (SCr) and blood urea nitrogen (BUN) levels in I/R injury. (B) Histological changes (H and E staining). Bar = 100 μm. (C) mRNA expression of Six1 in the I/R injured kidney was assessed by qRT-PCR. (D) Western blotting analysis of Six1 expression in the I/R injured kidney. (E) Immunofluorescence analysis of Six1 in IRI 2 days kidneys or sham. Bar = 50 μm. (F) Immunohistochemistry analysis of Six1 in IRI 2 days kidneys or sham. Bar = 50 μm. Data are mean ± SD for groups of six mice. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 versus sham ( t -test); # p <0.05 versus IRI 2 days (Bonferroni correction, two comparisons were made). IRI 1d, 1 day after IRI; IRI 2d, 2 days after IRI; IRI 3d, 3 days after IRI; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

Article Snippet: Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Immunohistochemistry

Journal: Frontiers in Molecular Biosciences

Article Title: SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice

doi: 10.3389/fmolb.2021.725319

Figure Lengend Snippet: Mouse Six1 suppresses monocyte chemotactic protein-1 ( MCP-1 ) expression by inhibiting NF-κB activation. (A) pNFκB-TA-luc reporter containing four NFκB binding sites (top). pNFκB-TA-luc co-transfected with pRL-SV40-N (internal control reporter plasmid) in mouse Six1 overexpression HK2 cells (HK2-Six1 6#) and the control 1#. Cells were harvested 24 h after transfection or treatment with 25 ng/ml TNFα for another 24 h, and luciferase activity was measured (bottom). (B) Plasmids (pNFκB-TA-luc and pRL-SV40-N) were co-transfected into the mouse renal tubular epithelial cells Six1 knockout (TCMK1-Six1 −/− 5#) and the Control 1#. Luciferase activity was measured. The relative luciferase activity was quantified by adjusting it to the renilla luciferase activity and untreated control samples were normalized to 1.0. (C) mRNA expression of mouse Six1 , human NFκB subunit RELA , human cofactors genes of RELA ( AES and FUS ) were assessed by qRT-PCR in the HK2-Six1 6# and the Control 1# cell lines. (D, E) Western blotting was applied to SIX1, RELA, p -RELA, AES, and FUS in HK2-Six1 cell lines (4# and 6#) and the Control cell lines (1# and 2#). (F) SIX1 DNA binding compatible motif (ACCTGA) in AES and FUS gene promoter regions and chromatin immunoprecipitation (ChIP) analysis of SIX1 occupancy of the AES and FUS genes from HK2-Six1 6# and the control 1# cell lines. The location of each primer set compared to the transcription start site (TSS) is shown (left). IgG control samples were normalized to 1.0. (G, H) After treatment by 25 ng/ml TNFα for 24 h or no treatment, mRNA expression of human MCP-1 and mouse Mcp-1 assessed by qRT-PCR in HK2-Six1 6# and Control 1#, TCMK1-Six1 −/− 5# and Control 1#, respectively. Data are expressed as mean ± SD. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 versus the Control group ( t -test). GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

Article Snippet: Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Binding Assay, Transfection, Control, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Knock-Out, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation

Journal: Frontiers in Molecular Biosciences

Article Title: SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice

doi: 10.3389/fmolb.2021.725319

Figure Lengend Snippet: Schematic depicting the effects of Six1 activation in renal tubular epithelial cells (TECs) on cell proliferation/migration and anti-inflammation and underlying mechanism.

Article Snippet: Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol.

Techniques: Activation Assay, Migration