Journal: Microsystems & Nanoengineering

Article Title: Modular operation of microfluidic chips for highly parallelized cell culture and liquid dosing via a fluidic circuit board

doi: 10.1038/s41378-020-00216-z

Figure Lengend Snippet: a Live-cell fluorescence images (one image per four chambers) showing an overview of all the chambers after 3 days of culturing GFP-expressing HUVECs. b Live-cell fluorescence image of GFP-expressing HUVECs in chambers 6 and 7. c Fluorescence image of fixed HUVECs with the cell F-actin and nuclei stained with ActinRed and NucBlue, respectively. d HUVECs after seeding and subsequent monolayer formation. The cells were seeded at a high cell density (i) and confluent on day 1 (ii). The monolayer was still intact on day 3 (iii) but began to deteriorate on day 4 (iv). At this point, the medium in the supply vial was replaced. The cells showed signs of recovery on day 5 (v) as the monolayer started to reform. The red dots in (iv) and (v) mark the cells counted in the region of interest. e Cells in ten chambers were counted in regions where the monolayer had deteriorated on day 4 (98 h) and after 16 h of recovery (114 h). The black dots represent the mean cell count, and the error bars represent the standard deviation. The cell number increased by an average of 33% in these areas

Article Snippet: Live-cell images of the GFP-expressing HUVECs (Angio-Proteomie, USA) were taken with an EVOS FL cell imaging system using the GFP filter cube.

Techniques: Fluorescence, Expressing, Staining, Cell Counting, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells

doi: 10.3389/fcell.2021.671461

Figure Lengend Snippet: ER stress induces expression of the primary and mature forms of miR-494. Relative mRNA expression of ER stress responsive genes as measured using qRT-PCR. (A) spliced XBP1 and Total XBP1 and (B) DDIT3 (CHOP) mRNA levels in HUVECs treated with 5 μg/mL tunicamycin (TCN) over a time course. Relative expression of (C) mature miR-494 and (D) primary miR-494 (pri-miR-494) in HUVECs treated with TCN over a time course. Gene expression is normalized to GAPDH or U6 and mean fold change compared to vehicle control or time 0h is shown. Graphs are representative of one biological replicate from three independent replicates where values indicate mean ± standard deviation.

Article Snippet: For plasmid transfections, HUVECs were transfected using the Cytofect HUVECs transfection kit (Cat:TF200K, Cell Applications). pEGFP-ATF6 was a gift from Ron Prywes (Addgene plasmid # 32955) ( ).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells

doi: 10.3389/fcell.2021.671461

Figure Lengend Snippet: miR-494 is a negative regulator of ER stress in vitro . Relative mRNA expression of ER stress responsive genes as measured by qRT-PCR. (A,B) DDIT3 (CHOP), (C,D) spliced XBP1 in HUVECs treated with 10 μg/mL TCN 48h after transfection with (A,C) miR-494 mimic or (B,D) miR-494 inhibitor. Gene expression is normalized to GAPDH and mean fold changes compared to control treatments are shown. (E,F) Simple Western blot analysis of HUVECs transfected with miR-494 mimic or control (24 h) (E) or miR-494 inhibitor or control (F) followed by TCN (10 μg/mL) for 24 h. (G) Cell viability in HUVECs as treated in (A) Vertical dotted red line indicates non-adjacent lanes. Graphs are mean + SEM fold changes of biological replicates from n = 3 independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 by two-tailed Student’s T -test.

Article Snippet: For plasmid transfections, HUVECs were transfected using the Cytofect HUVECs transfection kit (Cat:TF200K, Cell Applications). pEGFP-ATF6 was a gift from Ron Prywes (Addgene plasmid # 32955) ( ).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Gene Expression, Control, Simple Western, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells

doi: 10.3389/fcell.2021.671461

Figure Lengend Snippet: miR-494 regulates target genes in cell survival and DNA replication. (A) Venn diagram showing the number of downregulated target proteins in a Tandem Mass Tag labeled Mass Spectrometry profile from HUVECs treated with TCN or transfected with miR-494 compared to vehicle treatment or control miR respectively. (B) Fold-change (compared to respective controls) of protein or mRNA levels as assessed by Mass Spectrometry or qRT-PCR respectively for the six targets that were downregulated in both TCN and miR-494 groups. All six targets harbor miR-494 binding sites in their 3′UTRs. (C) Representative Simple Western blot showing survivin ( BIRC5 ) and GINS4 levels in HUVECs 24 h after miR-494 transfection followed by TCN treatment (24 h). Right panels show quantitation of biological replicates. ∗ P < 0.05, ∗∗ P < 0.01, by two-tailed Student’s T -test. (D) Immunofluorescence images showing survivin expression in HUVECs 24 h after miR transfection and/or TCN treatment. Right panel shows quantification via ImageJ of survivin fluorescence intensity in each cell. Each dot represents individual cells. Scale bar in white = 50 μm. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 by one-way ANOVA with post hoc Tukey’s correction.

Article Snippet: For plasmid transfections, HUVECs were transfected using the Cytofect HUVECs transfection kit (Cat:TF200K, Cell Applications). pEGFP-ATF6 was a gift from Ron Prywes (Addgene plasmid # 32955) ( ).

Techniques: Labeling, Mass Spectrometry, Transfection, Control, Quantitative RT-PCR, Binding Assay, Simple Western, Quantitation Assay, Two Tailed Test, Immunofluorescence, Expressing, Fluorescence