Journal: bioRxiv

Article Title: Notch Regulates Vascular Collagen IV Basement Membrane Through Modulation of Lysyl Hydroxylase 3 Trafficking

doi: 10.1101/2020.06.22.165431

Figure Lengend Snippet: (A) Representative images of ECs expressing RFP-Rab10 WT and stained for Col IV (green). Green arrowheads indicate CIVCs only and yellow arrowheads indicate Rab10 puncta only. (B) Representative images of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs and stained for Col IV (red), actin (green), and DNA (blue). Arrowheads denote extracellular Col IV secretion. (C) Graph of extracellular Col IV ratio of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs cultured in SS media. (D) Representative images of fibrin-bead sprouts between indicated siRNA treatment groups. Sprouts were stained for actin (grey) to delineate sprout morphology. (E-G) Graphs of sprouting parameters for scramble, Rab10, Rab25, or LH3 siRNA-treated sprouts. (H) Representative images of ECs cultured in VEGF-containing or SS media and stained for Col IV (green), LH3 (red), and DNA (blue). Green arrowheads indicate CIVCs only and yellow arrowheads indicate co-localized puncta. (I) Graph of percent CIVC vesicles co-localized with LH3 in ECs cultured in VEGF-containing or SS media. (J) Representative images of scramble, Rab10, Rab25, or both Rab10/25 siRNA-treated ECs and stained for Col IV (green), LH3 (red), actin (grey), and DNA (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV only puncta, and red arrowheads indicate LH3 puncta only. (K) Graph of percent CIVC vesicles co-localized with LH3 in scramble, Rab10, Rab25, or LH3 siRNA-treated conditions cultured in SS media or SS media with CLQ (10µM). (L) Representative images of ECs expressing RFP-Rab10 WT, CA, DN, stained for Col IV (green) and LH3 (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV puncta, and red arrowheads indicate LH3 puncta. (M) Graph of percent CIVC vesicles co-localized with LH3 in ECs expressing RFP-Rab10 WT, CA or DN. (N) Graph of percent Rab10 puncta co-localized with Rab25 in either VEGF containing or SS media. (O) Graph of percent Rab10 puncta co-localized with Rab25 in ECs transfected with indicated constructs. (P) Representative images of WT or Rab10 +/- mice retinas harvested at P6. Retinas were stained for Col IV (red), LH3 (blue) and IB4 (green) to identify blood vessels. (Q) Graph of percent CIVCs containing LH3 in WT or Rab10 +/- P6 mouse retinas. (R) Schematic diagram showing how Rab10 and Rab25 function to coordinate delivery of LH3 to CIVCs for proper secretion of Col IV. Trans-Golgi network (TGN). For all experiments, data are represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p-values. All p-values are from two-tailed Student’s t-test from at least three experiments. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns, not significant.

Article Snippet: Full-length human Rab25 cDNA was purchased from Origene (RC203413, ORIgene) and cloned into pME-MCS as described supplementary table 1.

Techniques: Expressing, Staining, Cell Culture, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Notch Regulates Vascular Collagen IV Basement Membrane Through Modulation of Lysyl Hydroxylase 3 Trafficking

doi: 10.1101/2020.06.22.165431

Figure Lengend Snippet: (A) Representative images of ECs co-expressing RFP-Rab10 WT and BFP-Rab25 WT in VEGF containing either VEGF-containing or SS media Yellow arrowheads denote co-localized Rab10 and Rab25 puncta, red arrowheads denote Rab10 puncta only, and green arrowheads denote Rab25 puncta only. (B) Representative images of ECs transfected to co-expression RFP-Rab10 WT, CA or DN and BFP-Rab25 WT, CA or DN cultured in SS media. Yellow arrowheads denote co-localized Rab10 and Rab25 puncta, red arrowheads denote Rab10 puncta only, and green arrowheads denote Rab25 puncta only. (C) Representative images of scramble and Rab25 siRNA-treated ECs cultured in SS media and stained for Col IV (grey). Dotted red and green lines indicates CIVC vesicle positive or negative ECs, respectively. Arrowheads denote CIVC vesicles. (D) Graph of number of ECs containing CIVC vesicles in scramble, Rab10, Rab25, and LH3 siRNA-treated ECs cultured in SS media. (E) Representative images of ECs transfected with either scramble or Vps33b siRNA and stained for Col IV (red), actin (green), and DNA (blue). Dotted green line indicates cell outline. Arrowheads denote extracellular Col IV secretion. (F) Graph of extracellular Col IV ratio of indicated siRNA treated ECs. n, number of measurements. (G) Representative images of ECs transfected with either scramble or Vps33b siRNA and stained for Col IV (red), LH3 (green), actin (light blue), and DNA (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV only puncta, and red arrowheads indicate LH3 puncta only. (H) Graph of percent CIVCs containing LH3 in scramble or Vps33b-siRNA treated ECs. For all experiments, data are represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p-values. All p-values are from two-tailed Student’s t-test from at least three experiments. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns, not significant.

Article Snippet: Full-length human Rab25 cDNA was purchased from Origene (RC203413, ORIgene) and cloned into pME-MCS as described supplementary table 1.

Techniques: Expressing, Transfection, Cell Culture, Staining, Two Tailed Test

Journal: Virulence

Article Title: MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells

doi: 10.1080/21505594.2020.1735051

Figure Lengend Snippet: Sequences of primers, miRNAs, and siRNAs used in this study.

Article Snippet: The membrane was blocked with the polyclonal anti-Rab25 antibody (CSB-PA019175LA01HU, cusabio), and monoclonal anti-β-actin antibody (AB0035, Abways).

Techniques: Sequencing, Over Expression, Knockdown