Journal: Oxidative medicine and cellular longevity

Article Title: Trehalose Protects Keratinocytes against Ultraviolet B Radiation by Activating Autophagy via Regulating TIMP3 and ATG9A.

doi: 10.1155/2022/9366494

Figure Lengend Snippet: Figure 1: Pretreatment of trehalose induces autophagy and inhibits migration of HaCaT cells. Western blot showing LC3A/B levels in 50 mJ/cm2 UVB-irradiated HaCaT cells treated (a, c) or pretreated (b, d) with or without 100 mM trehalose for 12, 24, or 48 hours in the absence (a, b) or presence (c, d) of 50 μM chloroquine. The statistical differences and fold increases of LC3-II/GAPDH ratios were calculated between cells treated with and without trehalose. Afterwards, HaCaT cells were pretreated with or without 100 mM trehalose for 24 hours before exposure to 50 mJ/cm2 UVB radiation or not and incubated for 12 hours. (e, f) BrdU and CCK-8 results showing cell proliferation ability. (g) Representative pictures of western blot showing PARP/cleaved PARP and caspase-3/cleaved caspase-3 expression levels. (h) Representative dot plot graphs generated from flow cytometric analysis showing the percentage of apoptotic cells. (i) Representative pictures of calcein AM staining of HaCaT cells in the Cell Migration Assay plate, and the original magnification is ×10. The percentage of cells invaded in the invasion zone was calculated to represent the cell migration rate. ∗∗∗P ≤0:001, ∗∗P ≤0:01, and ∗P ≤0:05. Con: control; h: hour (s); Q: quadrant; NS: no significance; Pre: pretreatment; TRE: trehalose.

Article Snippet: Reagents used in this study include chloroquine diphosphate (Life technologies, P36235), D-(+)-trehalose dehydrate (Sigma-Aldrich, T0167), acridine orange (AO) (Sigma-Aldrich, A9231), calcein AM (Dojindo Laboratories, C326), LysoTracker Deep Red (ThermoFisher Scientific, L12492), Mito-Tracker Green (Beyotime Biotechnology, C1048), ER-Tracker Red (Beyotime Biotechnology, C1041), DAPI Staining Solution (Beyotime Biotechnology, C1005), and TRIzol Reagent (Invitrogen, 15596026).

Techniques: Migration, Western Blot, Irradiation, Incubation, CCK-8 Assay, Expressing, Generated, Staining, Cell Migration Assay, Control

Journal: Oxidative medicine and cellular longevity

Article Title: Trehalose Protects Keratinocytes against Ultraviolet B Radiation by Activating Autophagy via Regulating TIMP3 and ATG9A.

doi: 10.1155/2022/9366494

Figure Lengend Snippet: Figure 4: TIMP3 and ATG9A contribute to trehalose-induced autophagy and cell death in UVB-irradiated HaCaT cells. (a) Western blot results showing TIMP3 and ATG9A expression levels in siRNA transfected HaCaT cells. HaCaT cells transfected by TIMP3/ATG9A siRNA or not were pretreated with 100 mM trehalose for 24 hours before exposure to 50 mJ/cm2 UVB radiation and incubated for 12 hours. (b) Western blot results showing LC3A/B and MMP9 expression levels. Ratios of LC3-II/GAPDH and MMP9/GAPDH were calculated. (c) Western blot results showing LC3A/B levels. Ratios of LC3-II/GAPDH were calculated. (d) Representative images of AO staining. Scale bar, 20 μm. Red/green fluorescence ratios were calculated to display autophagy levels. (e) Representative images of RFP-GFP- LC3B puncta analysis. Scale bar, 20 μm. The ratios of positive cells with yellow fluorescence were calculated to display autophagy levels. (f) LDH results showing the percentage of cell death. (g) BrdU results showing DNA synthesis levels. (h) CCK-8 results showing cell proliferation capacity. (i) Representative pictures of calcein AM staining of HaCaT cells in the Cell Migration Assay plate, and the original magnification is ×10. The percentage of cells invaded in the invasion zone was calculated to represent the cell migration rate. ∗∗∗P ≤0:001, ∗∗P ≤0:01, and ∗P ≤0:05. Con: control; h: hour (s); NS: no significance; TRE: trehalose.

Article Snippet: Reagents used in this study include chloroquine diphosphate (Life technologies, P36235), D-(+)-trehalose dehydrate (Sigma-Aldrich, T0167), acridine orange (AO) (Sigma-Aldrich, A9231), calcein AM (Dojindo Laboratories, C326), LysoTracker Deep Red (ThermoFisher Scientific, L12492), Mito-Tracker Green (Beyotime Biotechnology, C1048), ER-Tracker Red (Beyotime Biotechnology, C1041), DAPI Staining Solution (Beyotime Biotechnology, C1005), and TRIzol Reagent (Invitrogen, 15596026).

Techniques: Irradiation, Western Blot, Expressing, Transfection, Incubation, Staining, DNA Synthesis, CCK-8 Assay, Cell Migration Assay, Migration, Control

Journal: Oxidative medicine and cellular longevity

Article Title: Trehalose Protects Keratinocytes against Ultraviolet B Radiation by Activating Autophagy via Regulating TIMP3 and ATG9A.

doi: 10.1155/2022/9366494

Figure Lengend Snippet: Figure 5: TIMP3 and ATG9A contribute to mediating the impact of trehalose on autophagy, cell death, and migration of A431 cells. (a) The mRNA levels of TIMP3 and ATG9A detected by RT-qPCR in normal (n = 8), AK (n = 8), and cSCC (n = 8) skin tissues. U6 served as the endogenous control. (b) Representative pictures of IHC staining of TIMP3 and ATG9A in normal, AK, and cSCC skin tissues. Scale bar, 100 μm. Enlarged images were included. The integrated optical density of TIMP3 and ATG9A of normal (n = 8), AK (n = 8), and cSCC (n = 8) skin tissue slides were obtained from eight visions of four slides. In following experiments, A431 cells were pretreated with 100 mM trehalose for 24 hours before exposure to 50 mJ/cm2 UVB radiation and then incubated for 4 hours. (c) Western blot results showing TIMP3, ATG9A, and LC3A/B expression levels. Ratios of LC3-II/GAPDH were calculated. (d) Representative images of AO staining. Red/green fluorescence ratios were calculated to display autophagy levels. Scale bar, 20 μm. (e) Representative images of RFP- GFP-LC3B punta analysis. Scale bar, 20 μm. The ratios of positive cells with yellow fluorescence was calculated to display autophagy levels. (f) LDH results showing the percentage of cell death. (g) Representative pictures of calcein AM staining of A431 cells in the Cell Migration Assay plate, and the original magnification is ×10. The percentage of cells invaded in the invasion zone was calculated to represent the cell migration rate. ∗∗∗P ≤0:001, ∗∗P ≤0:01, and ∗P ≤0:05. Con: control; h: hour (s); IOD: integrated optical density; NS: no significance; TRE: trehalose.

Article Snippet: Reagents used in this study include chloroquine diphosphate (Life technologies, P36235), D-(+)-trehalose dehydrate (Sigma-Aldrich, T0167), acridine orange (AO) (Sigma-Aldrich, A9231), calcein AM (Dojindo Laboratories, C326), LysoTracker Deep Red (ThermoFisher Scientific, L12492), Mito-Tracker Green (Beyotime Biotechnology, C1048), ER-Tracker Red (Beyotime Biotechnology, C1041), DAPI Staining Solution (Beyotime Biotechnology, C1005), and TRIzol Reagent (Invitrogen, 15596026).

Techniques: Migration, Quantitative RT-PCR, Control, Immunohistochemistry, Incubation, Western Blot, Expressing, Staining, Cell Migration Assay

Journal: Lab on a Chip

Article Title: Machine learning-driven single-cell phenotyping in size-controlled microenvironments via parallel deterministic droplet microfluidics

doi: 10.1039/d5lc00839e

Figure Lengend Snippet: Deterministic single-cell encapsulation and selective gelation in multisized microgels. (A) Simultaneous generation of gel-coated MSCs in two distinct microgel sizes immediately following cell encapsulation, crosslinking, and emulsion breaking. (i) Representative images of viable (calcein-positive) MSCs encapsulated in alginate-RGD microgels. Scale bar = 50 μm. (ii) Quantification of average microgel thickness surrounding single cells. (iii) Sphericity measurements of microgels based on confocal imaging. N = 32–50 gel-coated cells per group. (B) Viability (%) of gel-coated single cells after encapsulation. (C) Yield: number of gel-coated single cells produced per hour. (D) Purity analysis: (i) percentage of viable, gel-coated single cells in the total collected product. (ii) Distribution of the number of cells per microgel. Dotted lines represent the expected values based on the Poisson distribution for each microgel size (small grey dots: 20 μm, large: 50 μm channel widths), assuming gelation occurs in all droplets. For (B)–(D), n = 3 independent experiments. ** p < 0.01, **** p < 0.0001, n.s. = not significant. Statistical comparisons: Welch's one-way ANOVA with Dunnett's T3 multiple comparisons test for (A, ii); paired t -test for (B)–(D). Error bars in SD.

Article Snippet: Cell viability was assessed by staining with calcein AM (1 : 1000 dilution; Biotium) for 30 min at 37 °C, and gels were immobilized on poly- l -lysine coated slides (Sigma-Aldrich) for fluorescence imaging.

Techniques: Single Cell, Encapsulation, Emulsion, Imaging, Produced

Journal: Stem Cell Research & Therapy

Article Title: Interleukin-1β induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration

doi: 10.1186/s13287-018-1032-9

Figure Lengend Snippet: Effects of IL-1β in morphology and interaction between HUVECs and MSCs. Labeled MSCs with CellTracker™ Orange were seeded on HUVECs stained with Calcein AM and co-cultivated for 30 to 240 min. After a period of 60 min, MSCs attached to HUVECs and the morphology were still spherical but developed form of cytoplasmic offshoot. a After 60 min, MSC became flattened and adhered to HUVEC monolayer. IL-1β promoted adhesion (left, 60 min) and transendothelial migration abilities (right, 240 min) of MSCs. b After 180 and 240 min, MSCs extended long plasmic filopodia and integrated into the HUVEC monolayer. Orthogonal projections illustrate that MSCs inserted into HUVEC monolayer (left, 180 min) and formation of filopodia caused transendothelial migration (right, 240 min). Arrows indicate MSC migration through the HUVEC. Horizontal bar: XZ plane of confocal image stack; vertical bar: YZ plane of confocal image stack. Scale bar = 10 μm

Article Snippet: HUVEC monolayers were stained with 8 μM Calcein AM (Tocris, UK) in serum-free medium for 30 min at 37 °C, then gently washed three times with PBS.

Techniques: Labeling, Staining, Migration