Journal: Frontiers in Cell and Developmental Biology

Article Title: Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells

doi: 10.3389/fcell.2022.839109

Figure Lengend Snippet: Intermittent high glucose imparts mesenchymal character in endothelial cells both in vitro and in vivo . (A , B) Immunoblot analysis of HUVEC lysates collected from cells treated with differential high-glucose treatment conditions and probed for α-SMA ( A , n = 3), Slug ( A , n = 5), CD144 ( A , n = 5), and CD31 ( B , n = 5) along with their respective densitometry quantification analysis. (C) Immunofluorescence analysis and costaining of HUVEC exposed to intermittent high glucose for α-SMA and CD144. DAPI staining to visualize the nucleus is shown in blue ( n = 3). (D) Immunohistochemistry of tissue sections from BTBR WT and BTBR Ob/Ob mice stained for α-SMA and CD31 ( n = 3). DAPI staining is shown in blue. Images were acquired with Zeiss LSM 780 Microscope. White arrowheads indicate endothelial cells expressing both CD31 and α-SMA. Degree of colocalization was analyzed using the Coloc2 plugin of ImageJ and Pearson correlation coefficient values were plotted to indicate the level of colocalization between CD31 and α-SMA. Values represent the mean ± SD. ** p < .01, and *** p < .001, either by unpaired t -test for two groups or the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli test for comparisons of multiple groups.

Article Snippet: HUVEC (#CL002-2XT25, Himedia) were cultured in HiEndoXL TM Endothelial Cell Expansion medium (#AL517; Himedia) supplemented with 4% endothelial growth supplement, 3% fetal bovine serum (#RM1112, FBS; Himedia), and 1X penicillin streptomycin glutamine (#10378, PS; Gibco).

Techniques: In Vitro, In Vivo, Western Blot, Immunofluorescence, Staining, Immunohistochemistry, Microscopy, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells

doi: 10.3389/fcell.2022.839109

Figure Lengend Snippet: Intermittent hyperglycemia caused increased levels of H3K4me3 through upregulation of MLL2 and WDR82. (A – F) Immunoblot analysis of HUVEC lysates collected from cells treated with differential/intermittent high-glucose treatment conditions and probed for H3K4me3 ( A , n = 7), MLL2 ( B , n = 6), WDR82 ( C , n = 5), SET1A ( D , n = 5), MLL1 (CT) ( D , n = 6), Menin ( D , n = 3), WDR5 ( D , n = 6), SET1B ( E , n = 3), and MLL1 (NT) ( F , n = 3). (G) Immunohistochemistry of tissue sections from control and Ob/Ob mice stained for H3K4me3 and CD31 ( n = 6). Fluorescence intensity AU values per individual cell nucleus are indicated together with the mean. Total number of nuclei, n ≥ 150. (H , I) Immunoblotting for H3K4me3 (H) , WDR82 (H), and MLL2 (I) in cultured HUVEC exposed to intermittent high glucose with a high-glucose concentration of 15 and 25 mM ( n = 3). (J – L) Immunoblot analysis of rat aorta tissue lysates collected from cells treated with intermittent high-glucose treatment conditions and probed for H3K4me3 (J) , MLL2 (K) and WDR82 (L) along with their respective densitometry quantification. Values represent the mean ± SD. * p < .05, ** p < .01, and *** p < .001, either by unpaired t -test for two groups or the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli test for comparisons of multiple groups.

Article Snippet: HUVEC (#CL002-2XT25, Himedia) were cultured in HiEndoXL TM Endothelial Cell Expansion medium (#AL517; Himedia) supplemented with 4% endothelial growth supplement, 3% fetal bovine serum (#RM1112, FBS; Himedia), and 1X penicillin streptomycin glutamine (#10378, PS; Gibco).

Techniques: Western Blot, Immunohistochemistry, Control, Staining, Fluorescence, Cell Culture, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells

doi: 10.3389/fcell.2022.839109

Figure Lengend Snippet: Elevated H3K4me3 upon intermittent high-glucose exposure enriched in Jagged1 and Jagged2 promoter, thereby causing their expression and Notch activation in endothelial cells. (A , B) Immunoblot analysis of endothelial cell lysates collected from cells treated with intermittent, transient, and constant high glucose and probed for N1-ICD ( A , n = 3) and Hes1 ( B , n = 3). (C , D) ChIP-qPCR analysis of H3K4me3 enrichment on Jagged1 ( C , n = 4) and Jagged2 ( D , n = 3) gene promoters in HUVEC exposed to intermittent hyperglycemia. (E , F) Transcript-level expression of Jagged1 ( E , n = 4) and Jagged2 ( F , n = 4) measured through RT-qPCR technique in HUVEC challenged with an intermittent high-glucose condition. (G , H) Immunoblot analysis of HUVEC lysates collected from cells treated with differential high-glucose treatment conditions and probed for Jagged1 ( G , n = 5) and Jagged2 ( H , n = 4). (I , J) Human glomerular endothelial cells (HGMEC) were treated with intermittent high-glucose treatment conditions and immunoblotted for Jagged1 ( I , n = 3) and Jagged2 ( G , n = 3). (K – M) Immunoblot analysis of rat aorta tissue lysates collected from cells treated with intermittent high-glucose treatment conditions and probed for N1-ICD ( K , n = 3) Jagged1 ( L , n = 3), Jagged2 ( M , n = 3). Values represent the mean ± SD. * p < .05, ** p < .01, and *** p < .001, either by unpaired t -test for two groups or the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli test for comparisons of multiple groups.

Article Snippet: HUVEC (#CL002-2XT25, Himedia) were cultured in HiEndoXL TM Endothelial Cell Expansion medium (#AL517; Himedia) supplemented with 4% endothelial growth supplement, 3% fetal bovine serum (#RM1112, FBS; Himedia), and 1X penicillin streptomycin glutamine (#10378, PS; Gibco).

Techniques: Expressing, Activation Assay, Western Blot, ChIP-qPCR, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Elevated H3K4me3 Through MLL2-WDR82 upon Hyperglycemia Causes Jagged Ligand Dependent Notch Activation to Interplay with Differentiation State of Endothelial Cells

doi: 10.3389/fcell.2022.839109

Figure Lengend Snippet: Catalytic inhibition or siRNA-mediated knockdown of MLL blocked intermittent high-glucose-dependent mesenchymal switch of endothelial cells. (A) HUVEC were exposed to intermittent high-glucose treatment condition in combination with OICR-9429 and incubated for a total of 5 days followed by immunoblot analysis by probing for α-SMA and Slug along with densitometry quantification of the blots ( n = 4). (B) HUVEC were transfected with MLL2 specific siRNA followed by challenging with intermittent high glucose and incubation for a total of 5 days. Resultant cell lysate was immunoblotted and probed for α-SMA and Slug along with densitometry quantification of the blots ( n = 4). Values represent the mean ± SD. ** p < .01, and *** p < .001, by the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli test.

Article Snippet: HUVEC (#CL002-2XT25, Himedia) were cultured in HiEndoXL TM Endothelial Cell Expansion medium (#AL517; Himedia) supplemented with 4% endothelial growth supplement, 3% fetal bovine serum (#RM1112, FBS; Himedia), and 1X penicillin streptomycin glutamine (#10378, PS; Gibco).

Techniques: Inhibition, Knockdown, Incubation, Western Blot, Transfection