Journal: bioRxiv

Article Title: Mechanical, Biochemical, and Multicellular Effects on Vessel Network Morphometrics in a Microfluidic Vasculature-on-a-Chip

doi: 10.1101/2025.08.28.672831

Figure Lengend Snippet: a) Fluorescence images showing vessel network development over 7 days (images shown are for FB1:EC5). Red: RFP-HUVECs; green: GFP-normal human lung fibroblasts. b) Illustration of fluorescent microbeads perfused through MVN and sample fluorescence image of microbeads in MVN. c) Overlay fluorescence image of microbeads (blue) in the vessel network (red), with two insets. (i) Magnification of inset showing a vessel branch with no beads, suggesting perfusion did not reach that branch (white arrows). (ii) Magnification of inset with all vessel branches perfused by beads. Scale bar = 500 mm for a) to c). d) Beads leaked out of vessels frequently near branches with jagged edges. e) Vessel branches with smooth edges contained the beads without leakage. f) Localized vessel leak near a vessel opening. (Top: raw image of vessels; Bottom: red = vessels, green = beads).

Article Snippet: Red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs, cAP-0001RFP) and green fluorescent protein-expressing human adult lung fibroblasts (GFP-hLFs, cAP-0033GFP) were purchased from Angio-Proteomie (Boston, MA, USA) and cultured on T75 flasks coated with Quick Coating solution (cAP-01, Angio-Proteomie) according to manufacturer’s protocol.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Mechanical, Biochemical, and Multicellular Effects on Vessel Network Morphometrics in a Microfluidic Vasculature-on-a-Chip

doi: 10.1101/2025.08.28.672831

Figure Lengend Snippet: Morphometric analysis of fibroblast effects on MVN morphology: branched-based metrics. a) Fluorescence images of RFP-HUVECs forming MVNs in the middle channel. Scale bar = 500 mm. b) Line plots of average percent vessel area over time. Shaded regions represent standard deviations. c) Bar graph of average percent vessel area grouped by days in culture. Each data point represents percent vessel area of an individual device unit (n=10, 23, 26, 16, and 12 for EGM-2, FB1:EC5, FB5:EC5, FB-CM, and Ang-V50, respectively). d) Boxplot of individual vessel branch diameters (measurable, as defined in ) on Day 7. Red line is the median; box ranges from 25 th to 75 th percentiles. Each data point represents a single vessel diameter, collected from all the device units for each condition. e) Bar graph of number of diameter outliers (as defined in ) per device unit. For d) and e), n = 10, 23, 32, 16, and 12 for EGM-2, FB1:EC5, FB5:EC5, FB-CM, and Ang-V50, respectively. A few vessel units included in d) and e) were excluded from b) and c) because the prerequisite binary conversion using a global threshold failed to capture the true morphology of the vessel network in these units. For all graphs: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs, cAP-0001RFP) and green fluorescent protein-expressing human adult lung fibroblasts (GFP-hLFs, cAP-0033GFP) were purchased from Angio-Proteomie (Boston, MA, USA) and cultured on T75 flasks coated with Quick Coating solution (cAP-01, Angio-Proteomie) according to manufacturer’s protocol.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Mechanical, Biochemical, and Multicellular Effects on Vessel Network Morphometrics in a Microfluidic Vasculature-on-a-Chip

doi: 10.1101/2025.08.28.672831

Figure Lengend Snippet: Morphometric analysis of fibroblast effects on MVN morphology: void-based metrics. a) Temporal line plots and d) bar graph (grouped by days in culture) of void count per device unit. b) Temporal line plots and e) box plot (grouped by days in culture) of average void area per device unit. c) Temporal line plots and f) box plot (grouped by days in culture) of average void roundness per device unit. Shaded regions represent standard deviations. Each data point represents either total void count in a device unit for d), or the average void area or void roundness across all voids in a device unit for e) and f). g) Polar histograms of void angles over time. Angles measured from the horizontal x-axis as defined in . Angles between 90 to 180 degrees were converted to their supplementary angles. For a) to g): n = 10, 23, 26, 16, and 12 for EGM-2, FB1:EC5, FB5:EC5, FB-CM, and Ang-V50, respectively. h) Bar graph of percent perfusable vessel networks, calculated as number of perfusable MVN units divided by total tested MVN device units. Blue: perfusable units with leakage Red: perfusable units without leakage. For h): n = 10,18,29,12, and 12 for EGM-2, FB1:EC5, FB5:EC5, FB-CM, and Ang-V50, respectively. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs, cAP-0001RFP) and green fluorescent protein-expressing human adult lung fibroblasts (GFP-hLFs, cAP-0033GFP) were purchased from Angio-Proteomie (Boston, MA, USA) and cultured on T75 flasks coated with Quick Coating solution (cAP-01, Angio-Proteomie) according to manufacturer’s protocol.

Techniques:

Journal: PLoS ONE

Article Title: C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

doi: 10.1371/journal.pone.0025497

Figure Lengend Snippet: A. Immunoblots for RSK, C/EBPβ-phospho-Thr217, procaspase 8 and C/EBPβ were performed on C/EBPβ immunoprecipitates from activated primary human LMF lysates as described in . RSK and phosphorylated C/EBPβ were induced in activated LMF but decreased in activated LMF treated with the ERK1/2 inhibitor (10 µg for 24 hr) or with the C/EBPβ peptide (200 µg for 24 hr). Inactive procaspase 8 was associated with phosphorylated C/EBPβ in untreated, activated LMF, while active caspase 8 was associated with unphosphorylated C/EBPβ in activated LMF treated with the ERK1/2 inhibitor or with the C/EBPβ peptide. Human LMF expressed full-length C/EBPβ from the second AUG . β-Actin was used to correct for lung lysate input. We performed single analysis of the samples. Results from triplicate samples of two independent experiments are shown. B. The ERK1/2 inhibitor decreased the fold association of RSK with C/EBPβ (n: 6 per group; 0.07+/−0.01, P <0.0001), and the fold expression of C/EBPβ (n: 6 per group; 0.05+/−0.006, P <0.0001) and C/EBPβ-PhosphoThr266 (n: 6 per group; 0.33+/−0.078, P <0.0001) in activated, human lung fibroblasts. There was also an increased fold association between unphosphorylated human C/EBPβ and active caspase 8 (n: 6 per group; active caspase 8; 5.70+/−0.59, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited C/EBPβ expression (n: 6 per group; 0.12+/−0.01, P <0.0001), the phosphorylation of C/EBPβ-Thr266 (n: 6 per group; 0.17+/−0.03, P <0.0001), the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001) and increased the association of RSK with C/EBPβ (n: 6 per group; 0.24+/−0.08, P <0.0001). We performed single analysis of the samples. C. α-SMA and TGF-β were induced in activated LMF but the expression of these fibrogenic genes was inhibited by treatment with the ERK1/2 inhibitor (10 µg for 24 hr) or with the peptide (200 µg for 24 hr). β-Actin was used to correct for lung lysate input. Representative results from two independent studies. D. The ERK1/2 inhibitor decreased the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). The cell permeant Ac-KAla217VD-CHO peptide also inhibited the fold expression of α-SMA (n: 6 per group; 0.24+/−0.11, P <0.0001) and TGF-β1 (n: 6 per group; 0.14+/−0.02, P <0.0001). We performed single analysis of the samples. E. Annexin-V-PE binding in vivo in activated LMF was increased after treatment with the ERK1/2 inhibitor (20 µg for 8 hr) or with the peptide (200 µg for 24 hr). Values are the percentage of cells expressing annexin-V-PE binding as described in . Activated, human lung fibroblasts treated with the ERK1/2 inhibitor (n: 6; 66.33+/−5.68%, P <0.0001) or with the Ac-KAla217VD-CHO peptide (n: 6; 61.00+/−9.27%, P <0.0001) displayed increased percent annexin-V binding compared to control (n: 6; 4.15+/−0.94%). We performed single analysis of the samples. Results from triplicate samples of three independent experiments are shown.

Article Snippet: Primary human lung fibroblasts isolated from normal human lung parenchyma (Cell Applications; San Diego, CA) were cryopreserved at first passage and can be cultured and propagated at least 12 population doublings.

Techniques: Western Blot, Expressing, Phospho-proteomics, Binding Assay, In Vivo, Control

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Exhaled breath condensates from healthy children induce cell death of in vitro cultured cells by activation of apoptosis

doi: 10.5114/ada.2019.87087

Figure Lengend Snippet: A – The culture of human lung fibroblasts (HLF) in the phase contrast microscope after 4-hour incubation with double distilled water (DDW), used as a test control (upper picture) or with a representative EBC sample (bottom picture). B – The membranes of Human Apoptosis Array exposed to HLF lysates incubated for 4 h with DDW (left picture) or a representative EBC sample (right picture). Each pair of spots in white frames represents a respective target molecule. The numbers at the frames correspond to respective molecules on graph C: 1 – CD95/Fas, 2 – pro-caspase-3, 3 – cleaved caspase-3, 4 – SMAC/Diablo, 5 – Hsp27, 6 – Hsp70. The brightest spots correspond to reference markers (dashed line frames). C – Relative expressions of selected apoptosis pathway-related proteins with the highest signal density, calculated as the percent of reference spots

Article Snippet: The normal human lung fibroblasts (HLF), purchased from Cell Applications Inc. (San Diego, CA), passages 4 th to 8 th , and murine endothelial cell line C166 (ATCC ® CRL-2581TM) from American Type Culture Collection (ATCC, Manassas, VA), were used for in vitro studies.

Techniques: Microscopy, Incubation, Control