Journal: International Journal of Molecular Sciences

Article Title: Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma

doi: 10.3390/ijms22020667

Figure Lengend Snippet: Mitochondrial membrane potential (Δψm) changes, ATP production and mitophagy in explant human melanoma cultures with lower free zinc stores (M5), average free zinc stores (M10) and higher free zinc stores (M9) exposed to 0.5 μM zinc pyrithione during 72 h. Cells were exposed to external zinc pyrithione and ( A ) loss of Δψm measured by decreased red fluorescence of JC-1 was determined in at least 1000 cells visualized by fluorescence microscopy. Results represent means ± SD of at least three independent experiments. # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons. ( B ) ATP production was measured in cell lysates by ATP bioluminescent assay kit ( C ). Mitophagy-specific fluorescence (Mitophagy Detection Kit) in cells exposed to zinc pyrithione alone or together with autophagy inhibitor chloroquine was determined fluorimetrically. Results represent means ± SD of at least three independent experiments. * p < 0.05 significantly higher compared to the beginning of treatment, # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons.

Article Snippet: Control and zinc pyrithione-treated cells of explant human melanoma grown in 96-well plates with black bottom were washed with PBS and incubated in 100 nM Mitophagy Dye Solution (Mitophagy Detection Kit (Dojindo Laboratories, Kumamoto, Japan)) for 35 min. After removal of the culture medium and washing with PBS, mitophagy-specific fluorescence was evaluated using a cell scoring module of MetaXpress ® Image Acquisition and Analysis Software.

Techniques: Membrane, Fluorescence, Microscopy, ATP Bioluminescent Assay

Journal: Nature Communications

Article Title: Pancreatic tumor eradication via selective Pin1 inhibition in cancer-associated fibroblasts and T lymphocytes engagement

doi: 10.1038/s41467-022-31928-7

Figure Lengend Snippet: a Establishment and validation of CAFs via high expressions of both FAP-α and α-SMA. b Cellular uptake of Cy5-labeled formulations on Pan02 cells, NIH-3T3 cells, and CAFs, measured by flow cytometry ( n = 3 biologically independent experiments; error bars, mean ± SD). c Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, imaged by fluorescent microscopy. Scale bars are 20 μm. Three independent repetitions got similar results. d Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, quantified by flow cytometry ( n = 6 biologically independent samples; error bars, mean ± SD). Cytotoxicity on Pan02 cells ( e ) and CAFs ( f ) ( n = 3 biologically independent experiments; error bars, mean ± SD). g Western blotting detection of Pin1 and relative proteins from Pan02 cells after treating with PBS, AG17724 (0.5 μM), or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. h Quantitative analysis of the western blotting results from Pan02 cells (Three independent repetitions got similar results.). i Western blotting detection of Pin1 and relative proteins from CAFs treated with PBS, AG17724, or antiCAFs-DMS after treating with PBS, AG17724 (0.5 μM) or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. j Quantitative analysis of the western blotting results from CAFs (Three independent repetitions got similar results.). One-way analysis of variance followed by Turkey posttests. Source data are provided as a Source Data file.

Article Snippet: Anti-FAP-α antibody (MAB9727-100, 0.25 μg/mL as working concentration.) and anti-phospho-CDC2/CDK1 (Y15) antibody (AF888-SP, 0.2 μg/mL as working concentration.) were purchased from R&D SYSTEMS.

Techniques: Biomarker Discovery, Labeling, Flow Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: Virological characteristics of SARS-CoV-2 BA.2 variant

doi: 10.1101/2022.02.14.480335

Figure Lengend Snippet: a,h , Growth kinetics of SARS-CoV-2 variants. b , Fluorescence microscopy. GFP area of infected VeroE6/TMPRSS2 cells at 48 h.p.i were measured. Numbers in the panel indicate the number of GFP-positive cells counted. c , Plaque assay. Diameter of plaques (20 plaques per virus) are summarized. d,f , S-based fusion assay. The fusion activity (arbitrary units) is shown. e , Western blot. Left, representative blots of S-expressing cells. ACTB is an internal control. Right, the ratio of S2 to the full-length S plus S2 proteins. g , Fold increase of pseudovirus infectivity by TMPRSS2 expression. Assays were performed in quadruplicate ( a,g,h ), octuplicate ( a , most left) or triplicate ( d–f ) and data are the average ± s.d. Each dot indicates the result from an individual plaque ( c ) and an individual replicate ( e,g ). In b and c , raw data are shown in and . Statistically significant differences between BA.2 and other variants through timepoints were determined by multiple regression ( a,d,f,h ). Familywise error rates (FWERs) calculated using the Holm method are indicated in the figures. Statistically significant differences between BA.1 and BA.2 were determined by two-sided Mann–Whitney U -tests ( b,d ) or two-sided paired Student’s t -tests ( e ).

Article Snippet: To measure the surface expression level of TMPRSS2 , HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).

Techniques: Fluorescence, Microscopy, Infection, Plaque Assay, Single Vesicle Fusion Assay, Activity Assay, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Virological characteristics of SARS-CoV-2 BA.2 variant

doi: 10.1101/2022.02.14.480335

Figure Lengend Snippet: a, Fluorescence microscopy. GFP area of infected VeroE6/TMPRSS2 cells (m.o.i. 0.01) at 24, 48, and 72 h.p.i were measured. Higher-magnification views of the regions indicated by squares are shown at bottom. b , Plaque assay. c , Coculture of S-expressing cells with HEK293-ACE2/TMPRSS2 cells. Left, representative images of S-expressing cells cocultured with HEK293 cells (top) or HEK293-ACE2/TMPRSS2 cells (bottom). Nuclei were stained with Hoechst 33342 (blue). Right, the size distribution of syncytia (green). Numbers in the panel indicate the numbers of GFP-positive syncytia counted. Data are the average ± s.d. A statistically significant difference between BA.1 an BA.2 was determined by two-sided Mann–Whitney U -test. In a and b , summarized data are shown in and . Scale bars, 500 μm ( a ) or 200 μm ( c ).

Article Snippet: To measure the surface expression level of TMPRSS2 , HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).

Techniques: Fluorescence, Microscopy, Infection, Plaque Assay, Expressing, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Virological characteristics of SARS-CoV-2 BA.2 variant

doi: 10.1101/2022.02.14.480335

Figure Lengend Snippet: a , S expression on the cell surface. Representative histograms stained with anti-S1/S2 polyclonal antibody (left) and the summarised data (right) are respectively shown. The number in the histogram indicates MFI. Grey histograms indicate isotype controls. b , Binding affinity of SARS-CoV-2 S RBD to ACE2 by yeast surface display. Left, The percentage of the binding of the SARS-CoV-2 S RBD expressed on yeast to soluble ACE2 (left) and the summarised data (right) are respectively shown. c , TMPRSS2 expression on the cell surface. Left, representative histograms stained with anti-TMPRSS2 polyclonal antibody are shown. The number in the histogram indicates MFI. Grey histograms indicate isotype controls. Right, summarized data. Assays were performed in triplicate, and data are the average ± s.d. Each dot indicates the result from an individual replicate. A statistically significant difference between BA.1 an BA.2 was determined by two-sided unpaired Student’s t -test ( a ) or two-sided Mann–Whitney U -test ( b ).

Article Snippet: To measure the surface expression level of TMPRSS2 , HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).

Techniques: Expressing, Staining, Binding Assay, MANN-WHITNEY

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 2: The effect of stretch on EVs and endoplasmic reticulum (ER) stress in ASMCs. (A) Left to right: representative immunofluorescence images of CD63/HSPA5 in ASMCs acquired with confocal microscopy (×100 objective), the quantified CD63/HSPA5 puncta number inside ASMCs (ImageJ), and Pearson coefficient of the pixel-intensity correlation of CD63/HSPA5 in ASMCs (ImageJ, scale bar = 200 μm); (B) Protein expression of CD63/HSPA5 in ASMCs (WB); (C) Size distribution and quantified counts, as well as protein concentration of EVs in ASMCs (NTA and BCA). ER: endoplasmic reticulum; HSPA5: biomarker protein of ER stress; TUDCA: tauroursodeoxycholic acid, ER stress inhibitor; NTA, nanoparticle tracking analysis; BCA: bicinchoninic acid assay; data present as means ± SD, experiments were repeated three times (n = 3), **p < 0.01 compared with Static, ##p < 0.01 compared with Stretch

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Protein Concentration, Biomarker Discovery, Acid Assay

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 6: The effect of mechanical ventilation on secretion of EVs, ER stress, airway inflammation and injury in mouse models of VILI. (A) Representative images of lung tissue slides with hematoxylin and eosin (HE) or immunohisto- chemistry (IHC, HSPA5) staining (scale bar = 100 μm, arrows indicate alveolar collapse, arrowhead indicate HSPA5 expression). Healthy, MV, MV + TUDCA/GW4869 indicates mice breathed spontaneously, under 18 mL/kg mechanical ventilation (MV), MV with pretreatment of TUDCA/GW4869, respectively; (B) Protein expression of HSPA5 in lung tissue with IHC staining from C57BL/6 mice treated with different conditions, respectively; (C–E) Size distribution and quantified count, HSPA5, as well as protein concentration of EVs isolated from bronchoalveolar lavage fluid (BALF) of mice treated with different conditions; (F) Relative TGF-β1 and IL-10 secretion in BALF from mice treated with different conditions. Data present as means ± SD, experiments were repeated six times (n = 6), ** p < 0.01 compared with Healthy, #p < 0.05, ##p < 0.01 compared with MV

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunohistochemistry, Staining, Expressing, Protein Concentration, Isolation

Journal: Nature Communications

Article Title: Estrogen-related receptor γ causes osteoarthritis by upregulating extracellular matrix-degrading enzymes

doi: 10.1038/s41467-017-01868-8

Figure Lengend Snippet: Signaling upstream of ERRγ expression. a qRT-PCR analysis ( n ≥ 8) of IL-6 in chondrocytes treated with IL-1β and infected with 800 MOI of control virus (Ad-C) or the indicated MOI of Ad- Epas1 or Ad- Slc3918 . b , c Detection of IL-6 in chondrocytes treated with IL-1β (1 ng ml –1 ) by immunofluorescence microscopy ( b ) and Western blotting ( c ). d , e Western blot ( d ) and qRT-PCR ( e ) analysis of ERRγ, MMP3, and MMP13 in chondrocytes treated with IL-6 ( n = 10). MMP3 and MMP13 proteins in conditioned medium of chondrocyte cultures were detected by Western blotting. f qRT-PCR analysis ( n ≥ 8) of ERRγ mRNA levels in chondrocytes treated with IL-1β (left panel). qRT-PCR analysis ( n ≥ 8) of ERRγ mRNA levels in chondrocytes treated with IL-6 siRNA and exposed to IL-1β (middle panel). qRT-PCR analysis ( n ≥ 8) of ERRγ mRNA levels in chondrocytes treated with AG490 and exposed to IL-6 (right panel). Values are presented as means ± s.e.m. (* P < 0.05, ** P < 0.005, and *** P < 0.0005. One-way ANOVA)

Article Snippet: Additional immunostaining was performed using antibodies against ERRα (40 μg ml –1 ; sc-65715; Santa Cruz Biotech), MMP3 (4 μg ml –1 ; ab52915; Abcam), MMP13 (1:100 dilution; ab51072; Abcam), SOX9 (12 μg ml –1 ; ab185966; Abcam), and type II collagen (2 μg ml –1 ; MAB8887; Millipore).

Techniques: Expressing, Quantitative RT-PCR, Infection, Immunofluorescence, Microscopy, Western Blot

Journal: Nature Communications

Article Title: Estrogen-related receptor γ causes osteoarthritis by upregulating extracellular matrix-degrading enzymes

doi: 10.1038/s41467-017-01868-8

Figure Lengend Snippet: ERRγ directly upregulates MMP3 and MMP13 in chondrocytes. a The mRNA (left) and protein (right) levels of ERRγ, MMP3, and MMP13 in primary cultured chondrocytes infected with Ad-C (800 MOI) or the indicated MOI of Ad- Esrrg for 36 h ( n = 12). b Representative images showing immunostaining for MMP3, MMP13, SOX9, and type II collagen (Coll-II) in cartilage sections of mice obtained after 3 weekly IA injections with Ad-C or Ad- Esrrg ( n = 12 mice per group). c The ERRE sequences in the promoter regions of Mmp3 and Mmp13 are indicated. d Quantitative ChIP assays were performed, and the data are presented as fold-changes relative to each input ( n ≥ 4). e qRT-PCR ( n = 11) analyses of MMP3 and MMP13 expression in chondrocytes treated with IL-6 (100 ng ml –1 ) in the presence of the indicated concentrations of GSK5182, an inverse agonist of ERRγ. f Sham-operated or DMM-operated mice were IP-injected with GSK5182 and killed 8 weeks after the operation. Representative images of safranin-O staining and scoring of OARSI grade, osteophyte size, and subchondral bone plate thickness ( n = 10 mice per group). Values are presented as means ± s.e.m. (* P < 0.05, ** P < 0.005, and *** P < 0.0005). One-way ANOVA ( a , d , e ), two-tailed t test ( f ), and Mann–Whitney U test ( f ). Scale bar: 50 μm

Article Snippet: Additional immunostaining was performed using antibodies against ERRα (40 μg ml –1 ; sc-65715; Santa Cruz Biotech), MMP3 (4 μg ml –1 ; ab52915; Abcam), MMP13 (1:100 dilution; ab51072; Abcam), SOX9 (12 μg ml –1 ; ab185966; Abcam), and type II collagen (2 μg ml –1 ; MAB8887; Millipore).

Techniques: Cell Culture, Infection, Immunostaining, Quantitative RT-PCR, Expressing, Injection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: mTOR controls growth and internal architecture of human breast cancer spheroids

doi: 10.1101/2024.02.24.580871

Figure Lengend Snippet: (A) Images of T47D-tfLC3 spheroids 0, 24, 48 and 72 hours after treatment with rapamycin, bafilomycin and starvation (EBSS). (B) Quantitative evaluation of the GFP/RFP ration of T47D-tfLC3 spheroids over time. Data are normalized to GFP/RFP ratio at time point 0 hours. Error bars indicate SEM (n=18). (C) A representative image of a Western blot of T47D wild type spheroids after seven days of treatment. (D) Quantification of LC3 I to LC3 II turnover by relative densitometry. Data are normalized to the DMSO control. Error bars indicate SEM (n=4). (E) Western blot of T47D wild type and ATG7 knock-out spheroids. Microscope: Zeiss Cell Observer automated wide-field fluorescence microscope equipped with incubator box and environmental control system. (37 °C, 5% CO2). Images were recorded over 72 hours at an interval of 1 hour for each channel (GFP, mRFP) and each well.

Article Snippet: Membranes were blocked with fat-free dry milk in TBS-T. Primary antibodies against GAPDH (AM4300, Ambion), LC3 (NB100-2220, Novus Biologicals), P70-S6K1 and phophoP70-S6K1 (9202, 9206, Cell Signaling, Danvers), APG7 (Santa Cruz - sc-8668, n-20) were incubated over night at 4°C.

Techniques: Western Blot, Control, Knock-Out, Microscopy, Fluorescence