Journal: Current Issues in Molecular Biology

Article Title: Tauroursodeoxycholic Acid Confers Protection Against Oxidative Stress via Autophagy Induction in Retinal Pigment Epithelial Cells

doi: 10.3390/cimb47040224

Figure Lengend Snippet: TUDCA increases LC3-II protein expression. ARPE-19 cells were treated with 800 µM H 2 O 2 , 500 µM TUDCA, or H 2 O 2 + TUDCA for 24 h, and protein lysate was collected to measure LC3 and p62 expression. Untreated cells served as a control. Data of ( A ) LC3-II (n = 3) and ( B ) p62 (n = 2) protein expression represented as mean ± standard deviation. * p < 0.05 compared to control. **** p < 0.0001 compared to control. ( C ) Representative Western blots of LC3 and p62.

Article Snippet: Cells were fixed with 1:1 methanol/acetone for 10 min and blocked in 2% BSA in PBS + Tween20 (0.05%) for 1 h. Primary antibodies LC3 (1:200, CST #12741) and p62 (1:200, CST #88588) were added to the blocking buffer for 1 h at room temperature.

Techniques: Expressing, Control, Standard Deviation, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Tauroursodeoxycholic Acid Confers Protection Against Oxidative Stress via Autophagy Induction in Retinal Pigment Epithelial Cells

doi: 10.3390/cimb47040224

Figure Lengend Snippet: TUDCA and H 2 O 2 increase the colocalization of LC3 and p62. ( A ) ARPE-19 cells were ( a ) untreated; treated with ( b ) 200 µM H 2 O 2 , ( c ) 500 µM TUDCA, or ( d ) H 2 O 2 + TUDCA for 24 h and were stained for LC3 and p62 localization. ( B ) Quantification of colocalized LC3 and p62. * p < 0.05 *** p < 0.001 compared to control. Green puncta: LC3. Red puncta: p62. Yellow puncta: colocalization. Magnification, 60×. Scale bar, 20 µm.

Article Snippet: Cells were fixed with 1:1 methanol/acetone for 10 min and blocked in 2% BSA in PBS + Tween20 (0.05%) for 1 h. Primary antibodies LC3 (1:200, CST #12741) and p62 (1:200, CST #88588) were added to the blocking buffer for 1 h at room temperature.

Techniques: Staining, Control

Journal: Current Issues in Molecular Biology

Article Title: Tauroursodeoxycholic Acid Confers Protection Against Oxidative Stress via Autophagy Induction in Retinal Pigment Epithelial Cells

doi: 10.3390/cimb47040224

Figure Lengend Snippet: TUDCA increases autophagy flux. ARPE-19 cells were treated with 500 µM TUDCA, 100 nM bafilomycin, or bafilomycin + TUDCA for 3 h, and protein lysate was collected to measure LC3 expression. Untreated cells served as a control. Data of LC3-II ( A ) and LC3-II/LC3-I ( B ) protein expression represented as mean ± standard deviation (n = 2). ** p < 0.01 compared to control. *** p < 0.001 compared to control. † p < 0.05 compared to bafilomycin alone. ( C ) Representative Western blots of LC3.

Article Snippet: Cells were fixed with 1:1 methanol/acetone for 10 min and blocked in 2% BSA in PBS + Tween20 (0.05%) for 1 h. Primary antibodies LC3 (1:200, CST #12741) and p62 (1:200, CST #88588) were added to the blocking buffer for 1 h at room temperature.

Techniques: Expressing, Control, Standard Deviation, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Tauroursodeoxycholic Acid Confers Protection Against Oxidative Stress via Autophagy Induction in Retinal Pigment Epithelial Cells

doi: 10.3390/cimb47040224

Figure Lengend Snippet: TUDCA and H 2 O 2 increase the colocalization of LC3 and p62 in iPSC-derived RPE. ( A ) iPSC-derived RPE cells were ( a ) untreated, treated with ( b ) 200 µM H 2 O 2 , ( c ) 500 µM TUDCA, or ( d ) H 2 O 2 + TUDCA for 24 h and were stained for LC3 and p62 localization. ( B ) Quantification of colocalized LC3 and p62. *** p < 0.001 compared to control. Green puncta: LC3. Red puncta: p62. Yellow puncta: colocalization. Magnification, 60×. Scale bar, 20 µm.

Article Snippet: Cells were fixed with 1:1 methanol/acetone for 10 min and blocked in 2% BSA in PBS + Tween20 (0.05%) for 1 h. Primary antibodies LC3 (1:200, CST #12741) and p62 (1:200, CST #88588) were added to the blocking buffer for 1 h at room temperature.

Techniques: Derivative Assay, Staining, Control

Journal: Current Issues in Molecular Biology

Article Title: Tauroursodeoxycholic Acid Confers Protection Against Oxidative Stress via Autophagy Induction in Retinal Pigment Epithelial Cells

doi: 10.3390/cimb47040224

Figure Lengend Snippet: Effects of TUDCA treatment in iPSC-RPE. iPSC-RPE cells were treated with 500 µM TUDCA, 100 nM bafilomycin, or bafilomycin + TUDCA for 3 h, and protein lysate was collected to measure LC3 expression. Untreated cells served as a control. Data of LC3-II ( A ) and LC3-II/LC3-I ( B ) protein expression represented as mean ± standard deviation (n = 2). * p < 0.05 compared to control. ( C ) Representative Western blots of LC3.

Article Snippet: Cells were fixed with 1:1 methanol/acetone for 10 min and blocked in 2% BSA in PBS + Tween20 (0.05%) for 1 h. Primary antibodies LC3 (1:200, CST #12741) and p62 (1:200, CST #88588) were added to the blocking buffer for 1 h at room temperature.

Techniques: Expressing, Control, Standard Deviation, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Tauroursodeoxycholic Acid Confers Protection Against Oxidative Stress via Autophagy Induction in Retinal Pigment Epithelial Cells

doi: 10.3390/cimb47040224

Figure Lengend Snippet: TCA and TCDCA increase LC3-II protein expression. ARPE-19 cells were treated with 500 µM TCA, 500 µM TCDCA, 800 µM H2O2, H 2 O 2 + TCA, or H 2 O 2 + TCDCA for 24 h, and protein lysate was collected to measure LC3 and p62 expression. Untreated cells served as a control. Data of LC3-II ( A ) and p62 ( B ) protein expression represented as mean ± standard deviation (n = 3). * p < 0.05 compared to control. ** p < 0.01 compared to control. ( C ) Representative Western blots of LC3 and p62.

Article Snippet: Cells were fixed with 1:1 methanol/acetone for 10 min and blocked in 2% BSA in PBS + Tween20 (0.05%) for 1 h. Primary antibodies LC3 (1:200, CST #12741) and p62 (1:200, CST #88588) were added to the blocking buffer for 1 h at room temperature.

Techniques: Expressing, Control, Standard Deviation, Western Blot

Journal: Journal of pineal research

Article Title: Melatonin Increased Autophagy Level to Facilitate Osteogenesis of Inflamed PDLSCs Through TMEM110 Signaling Pathways.

doi: 10.1111/jpi.70039

Figure Lengend Snippet: FIGURE 4 | Melatonin restores damaged cell autophagy in an inflammatory microenvironment. (A) Fluorescence images of RFP‐GFP‐LC3 adenovirus‐infected Inf‐PDLSCs stimulated with melatonin at various concentrations. (B) TEM of ultrastructural changes in Inf‐PDLSCs. The yellow arrowheads indicate autophagosomes. (C) Quantitative analysis of the green and red spots number indicating RFP‐GFP‐LC3 adenovirus‐infected Inf‐ PDLSCs treated with gradient concentrations of melatonin (n = 4). (D) Quantitative analysis of autophagosomes number in Inf‐PDLSCs treated with gradient concentrations of melatonin (n = 4). (E) The LC3II/I ratio, (F) p62 expression and (G) Beclin1 expression in Inf‐PDLSCs treated with gradient concentrations of melatonin (n = 4). (H) The LC3II/I ratio of the Con and Mel groups with or without Bafilomycin A1 (Baf) treatment. *, **, and *** mean differences from Con group are statistically significant (p < 0.05, 0.01 and 0.001, respectively). #, ### mean differences from 0.5 μM group are statistically significant (p < 0.05 and 0.001). & & & means differences from 1 μM group are statistically significant (p < 0.001).

Article Snippet: To evaluate autophagy level, primary antibodies against LC3 I/II (CST, #12741), Beclin1 (CST, #3495), and p62 (CST, #88588) were used by Western blot analysis.

Techniques: Fluorescence, Infection, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses

doi: 10.3389/fcimb.2024.1334211

Figure Lengend Snippet: Nucleotide sequences (5’→3’) of primers used in qPCR.

Article Snippet: Cells were then incubated with rabbit anti-LC3 primary antibody (1:100, clone H50, Santa Cruz Biotechnology) for 1 h at 4°C, rinsed with permeabilization solution and incubated for 30 min with goat anti-rabbit secondary antibody conjugated with Alexa 488 (1:400, A-11059).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses

doi: 10.3389/fcimb.2024.1334211

Figure Lengend Snippet: Hydatid fluid from Echinococcus granulosus induces gene expression in the autophagy pathway in BMDCs. BMDCs (1×10 6 /ml) were cultured alone (CTRL), treated with 20 nM of Rapamycin (RAPA), with HF stimulation 200μg, or HF in the presence of rapamycin (HF+RAPA). Gene transcription of lc3, beclin 1, atg16l1 and atg12 , was analyzed 6 h post-stimulation from isolated mRNA by quantitative PCR (relative to the expression of GAPDH mRNA). Results are the mean ± SEM of four experiments performed in duplicate (one-way ANOVA test: ****p<0.0001, **p<0.01, and Tukey post hoc test ****p<0.0001, **p<0.01 for HF-treated BMDCs vs CTRL or HF+RAPA-treated BMDCs vs RAPA-treated BMDCs when is indicated).

Article Snippet: Cells were then incubated with rabbit anti-LC3 primary antibody (1:100, clone H50, Santa Cruz Biotechnology) for 1 h at 4°C, rinsed with permeabilization solution and incubated for 30 min with goat anti-rabbit secondary antibody conjugated with Alexa 488 (1:400, A-11059).

Techniques: Gene Expression, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses

doi: 10.3389/fcimb.2024.1334211

Figure Lengend Snippet: Hydatid fluid from Echinococcus granulosus induces accumulation of LC3 in a conjugated form on autophagosome membranes BMDCs (1×10 6 /ml) were cultured without stimulation (CTRL) and treated with 100 μM of chloroquine (CQ), stimulated with 200 μg of HF, or HF in the presence of chloroquine (HF+CQ). (A) Confocal images reveal the distribution of LC3 (green) and cell nuclei (blue) in BMDCs. Arrows highlight punctate LC3 structures in the cytoplasm. Scale bar 5 μm (B) Bar graphs show the number and MFI of LC3 + structures ± SEM of different cells in a representative experiment of three independent experiments (one-way ANOVA test and Tukey’s post hoc test, **p<0.01 and ****p<0.0001 HF-stimulated cells vs. CTRL or HF+CQ-treated BMDCs vs. CQ-treated BMDCs when indicated). (C) Cell permeabilization allows quantification of LC3 II-containing autophagosome fluorescence using Flow Cytometry. Zebra graphs shows percentage of intracellular LC3 + compartments of CTRL, HF-stimulated-BMDCs, treated with CQ alone or in combination with HF stimulation (HF+CQ) for 1 h. Negative control (NEG CTRL) represents the specificity of sample labeling when incubated with the secondary antibody in the absence of the primary anti-lc3. (D) Mean intensity fluorescence (MFI) under the same experimental conditions (one-way ANOVA test and Tukey’s post-hoc test, **p<0.01 and ***p<0.001 HF-stimulated cells vs. CTRL or HF+ CQ-treated BMDCs vs. CQ-treated BMDCs when indicated).

Article Snippet: Cells were then incubated with rabbit anti-LC3 primary antibody (1:100, clone H50, Santa Cruz Biotechnology) for 1 h at 4°C, rinsed with permeabilization solution and incubated for 30 min with goat anti-rabbit secondary antibody conjugated with Alexa 488 (1:400, A-11059).

Techniques: Cell Culture, Fluorescence, Flow Cytometry, Negative Control, Labeling, Incubation

Journal: JOR spine

Article Title: Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.

doi: 10.1002/jsp2.1286

Figure Lengend Snippet: FIGURE 1 Effect of bafilomycin-mediated autophagy flux inhibition in NP cells in vitro. (A) Experimental design for the bafilomycin treatment in rat NP cell culture. Rat NP cells were treated with Bafilomycin A1 in high nutrients (HN) and low nutrients (LN) media for indicated time and analyzed for different measures. (B) Expression of two common autophagy markers LC3-II and SQSTM1/p62 as determined by western blot following baf A1 treatment for 8–48 h in HN and LN media. (C) Cellular morphology of NP cells in LN media with baf (10 nM, 8–48 h) treatment was visualized using light microscopy. Cellular metabolism of NP cells in LN media with 10 nM baf treatment (8–48 h) was quantified using CCK-8 assay. (D) Representative images of live NP cells (stained green) and dead NP cells (stained red) following incubation in LN media +/ baf (8–48 h) treatment. The ratio of live (green)/ dead (red) cells in LN media +/ 10 nM baf treatment was quantified using image J. Data shown are mean ± SEM of 3 technical replicates. Two-tailed Student's t test was used to quantify significance. *p < 0.05 baf A1, bafilomycin A1.

Article Snippet: Primary antibodies to LC3 (12741, cell signaling technology), ATG7 (8558, cell signaling technology), ATG12-ATG5 (4180, cell signaling technology), SQSTM1/p62 (5114, Cell Signaling Technology), TNF-α (3707, cell signaling technology), IL-1β (12 242, cell signaling technology), p53 (2524, cell signaling technology), GAPDH (2118, cell signaling technology) and β-actin (A2066, Millipore Sigma), and secondary anti-rabbit HRP antibody (31460, Thermo Fisher) were used.

Techniques: Inhibition, In Vitro, Cell Culture, Expressing, Western Blot, Light Microscopy, CCK-8 Assay, Staining, Incubation, Two Tailed Test

Journal: JOR spine

Article Title: Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.

doi: 10.1002/jsp2.1286

Figure Lengend Snippet: FIGURE 5 Successful inhibition of autophagy in NP tissue. Autophagy was inhibited primarily in NP tissue of Col2-Cre; Atg7fl/fl mice. (A) Expression of ATG7, p62, LC3-II, and ATG12-ATG5 protein in AF and NP tissue from Atg7fl/fl control and Col2-Cre; Atg7fl/fl experimental mice at 3, 6, and 12 months (mo) of age was determined by Western blotting. β-actin was used as an internal loading control. (B) Densitometric analysis of the proteins in AF and NP tissue were quantified by dividing the amount of each protein by β-actin and normalizing to control protein amount for each age group. Data shown are mean ± SD of 3 independent mice per tissue per age group. Two-tailed unpaired t test was used to quantify significance and p-values are shown in the graphs. fl/fl, Atg7fl/fl, KO or Atg7 KO, Col2-Cre; Atg7fl/fl.

Article Snippet: Primary antibodies to LC3 (12741, cell signaling technology), ATG7 (8558, cell signaling technology), ATG12-ATG5 (4180, cell signaling technology), SQSTM1/p62 (5114, Cell Signaling Technology), TNF-α (3707, cell signaling technology), IL-1β (12 242, cell signaling technology), p53 (2524, cell signaling technology), GAPDH (2118, cell signaling technology) and β-actin (A2066, Millipore Sigma), and secondary anti-rabbit HRP antibody (31460, Thermo Fisher) were used.

Techniques: Inhibition, Expressing, Control, Western Blot, Two Tailed Test