Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Genes related to the excretion GO term and upregulated by NMDA injection and downregulated by KUS121 and KUS187 treatment.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Injection

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: mRNA expressions of endothelin-1 ( Edn1 ), endothelin receptor type A ( Ednra ) and endothelin receptor type B ( Ednrb ) in the mouse retina and primary retinal ganglion cells (RGCs). ( a – c ) Relative mRNA expression in neural retina. Neural retina of non-treated wild-type mice (W, n = 7) and NMDA-injected (C, n = 8), NMDA-injected-KUS121-treated (K121, n = 8) and NMDA-injected-KUS187-treated (K187, n = 8) mice were analyzed. The relative expression levels of Edn1 ( a ), Ednrb ( b ) and Ednra ( c ) mRNA were analyzed using qRT-PCR. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. * p < 0.05 and ** p < 0.01, vs. W, Tukey’s honestly significant difference (HSD). NS no significant difference compared with W, Tukey’s HSD. ( d – f ) Relative mRNA expression in primary RGCs. Primary RGCs isolated from 3-day-old rats by two-step immunopanning were cultured with or without KUS121 (50 µM) for 2 h and then with or without KUS121 and with or without NMDA (500 µM) for another 4 h. The relative expression levels of Edn1 ( d ), Ednrb ( e ) and Ednra ( f ) mRNA were analyzed using qRT-PCR. (-): without KUS121 without NMDA n = 3, C: with NMDA without KUS121, n = 3, K121: with NMDA with KUS121, n = 3, respectively. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. ** p < 0.01, control vs. KUS121, NS no significant difference compared with (-), Tukey’s HSD.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Injection, Quantitative RT-PCR, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Protein expression of endothelin-1 (EDN1) and endothelin receptor type B (EDNRB) in retinal tissue. ( a ) The retina of non-treated wild-type (labeled ‘‘W’’), NMDA-injected (control, labeled ‘‘C’’), or NMDA-injected-KUS121-treated mice (labeled ‘‘K’’) was analyzed by western blotting using an anti-EDN1 antibody. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. a. ( b ) Comparison of EDN1 expression shown as ratios to actin ( n = 5, for all treatments). ** p < 0.01, vs. W, Tukey’s HSD. ( c – f ) Vertical sections of non-treated wild-type, NMDA-injected, NMDA-injected-KUS121-treated and NMDA-injected-KUS187-treated mice retinae were stained with an anti-EDN1 antibody or anti-EDNRB antibody. ( c , d ) Staining intensities of RGC layers with anti-EDN1 ( c ) or anti-EDNRB ( d ) antibody. The staining intensity of the RGC layer at distances of 400–800 μm from the optic nerve head was analyzed using BZ II Analyzer software. W: non-treated wild-type, C: NMDA-injected, K121: NMDA-injected-KUS121-treated, K187: NMDA-injected-KUS187-treated. ( n = 3, for C and K121, n = 2, for W and K187) NS no significant difference compared with W, Tukey’s HSD. ( e,f ) Vertical sections of non-treated wild-type (WT), NMDA-injected (control), NMDA-injected-KUS121-treated (K121) and NMDA-injected-KUS187-treated mice (K187) retinae. The black bar represents 100 µm. GCL ganglion cell layer, IPL inner plexiform layer.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Labeling, Injection, Western Blot, Staining, Software

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: EDN1 and EDNRB protein expression and cell viability in 661W cultured cells under glucose-free conditions. ( a – d ) The relative amount of live cells was measured using WST-8 following 48 h treatment with DMEM/high glucose media or DMEM/glucose-free media with or without KUS121 (100 µM). ( a ) Quantitative analysis of live cells ( n = 3, for all treatments). 661W cells were cultured with high glucose [labeled ‘‘(-)’’] or treated in glucose-free media with (labeled ‘‘K121’’) or without (control, labeled ‘‘C’’) KUS121. ( b – d ) Representative images of 661W cells cultured under each condition. The black bar represents 50 µm. ( e – h ) Protein expression of EDN1 ( e , g ) and EDNRB ( f , h ) in 661W cells was analyzed by western blotting. 661W cells were cultured with high glucose media [labeled ‘‘(-)’’] or with glucose-free media with (labeled ‘‘K’’) or without (control, labeled ‘‘C’’) KUS121 for 24 h before the analysis. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. b,c. Relative expression of EDN1 ( g ) and EDNRB ( h ) was shown as a ratio to actin ( n = 4, for both treatments). * p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the control (in a and g ) or to the cells cultured in high glucose media (in h ), Tukey’s HSD.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Labeling, Western Blot

Journal: iScience

Article Title: Quantitative proteomics and RNA-sequencing of mouse liver endothelial cells identify novel regulators of BMP6 by iron

doi: 10.1016/j.isci.2023.108555

Figure Lengend Snippet:

Article Snippet: Total RNA was isolated from FACS-sorted endothelial cells using PicoPure RNA Isolation Kit (Applied Biosystems).

Techniques: Recombinant, SYBR Green Assay, Staining, TUNEL Assay, Isolation, Peptide Fractionation, DC Protein Assay, Negative Control, Software