Journal: Cells

Article Title: LncRNA NR_003508 Boosts Pseudomonas aeruginosa -Induced Autophagy by Facilitating the Conversion of LC3-I to LC3-II and p62 Degradation

doi: 10.3390/cells15141306

Figure Lengend Snippet: PA infection promotes autophagy. A volcano plot ( A ) and heatmap ( B ) illustrated the differentially expressed genes when comparing the PA group to the control group. Analyses of the differentially expressed genes include Gene Ontology (GO) ( C ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ( D ) studies. ( E ) The expression differences of autophagy-related genes were displayed on KEGG graph rendered by Pathview (Version 1.46.0). By superimposing the transcriptome differential expression data, the macroautophagy regulatory network with mTOR as the core switch intuitively displayed the expression trend of key autophagy genes in this experimental sample. ( F ) The expression levels of LC3 and p62 in C57BL/6J lung tissue infected with PA at different time points were assessed using Western blot analysis. ( G ) The gray value ratios of LC3II/LC3I and p62/β-actin ( n = 3). * p < 0.05, ** p < 0.01 vs. the control cells. ( H ) IHC was used to observe the expression of LC3 in mouse lung tissue by intratracheal instillation of PA at varied time points. The tissue sections were structurally intact. After PA infection, there was marked cell recruitment (nuclei blue) and a significant increase in the number of LC3-positive cells (positive signal was mainly brown-yellow to tan) at 3 and 6 h. The data were based on at least three independent experiments presented as mean ± SD ( n = 3, each group).

Article Snippet: The primary antibodies β-actin (Proteintech, Chicago, IL, USA; 66009-1-Ig; Dilution multiple 1:2000), Atg5 (Proteintech, Chicago, IL, USA; 10181-2-AP; Dilution multiple 1:1000), Atg7 (Proteintech, Chicago, IL, USA; 67341-1-Ig; Dilution multiple 1:1000), Atg12 (Abclonal, Wuhan, China; A22788; Dilution multiple 1:1000), LC3 (Abclonal, Wuhan, China; A12319; Dilution multiple 1:2500), and p62 (Proteintech, Chicago, IL, USA; 66184-1-Ig; Dilution multiple 1:2000) were utilized for the Western blot, along with HRP-conjugated Goat Anti-Rabbit/Mouse IgG(H + L) (Proteintech, Chicago, IL, USA; SA00001-2/-1; 1:5000 dilution).

Techniques: Infection, Control, Expressing, Quantitative Proteomics, Western Blot

Journal: Cells

Article Title: LncRNA NR_003508 Boosts Pseudomonas aeruginosa -Induced Autophagy by Facilitating the Conversion of LC3-I to LC3-II and p62 Degradation

doi: 10.3390/cells15141306

Figure Lengend Snippet: LncRNA NR_003508 accelerates autophagy with PA infection. ( A , E ) The LC3 and p62 expression levels with varying doses of PA infection were detected by Western blot in RAW264.7 and BMDM. ( C , G ) The LC3 and p62 expression infected with PA for varied time points were detected by Western blot in RAW264.7 and BMDM. ( B , D , F , H ) The gray value ratios of LC3II/LC3I and p62/β-actin were computed to create the line chart ( n = 3). * p < 0.05, ** p < 0.01 vs. the control cells. ( I ) Figure of the change of autophagy rate in RAW264.7 cells following transfection with si- NR_003508 by flow cytometry. ( J ) Quantitative analysis of autophagy rate. * p < 0.05 vs. PA, ** p < 0.01 vs. NC. In ( I ), the X-axis represented the fluorescence intensity of autophagy positive markers, and the Y-axis paired the number of cells under the fluorescence intensity. The area of autophagy-positive cells was defined by M1 gate, and the left side of M1 gate was autophagy-negative cells; that is, autophagy rate = M1-positive cells ÷ total cells × 100%. ( K ) The change in autophagy flux in RAW264.7 cells following transfection with si- NR_003508 was assessed using mRFP-GFP-LC3 (scale = 2.5 μm). ( L ) Quantitative analysis of autophagy flux. * p < 0.05 vs. PA, ** p < 0.01 vs. NC. The representativeness of three independent experiments was shown as mean ± SD ( n = 3, each group).

Article Snippet: The primary antibodies β-actin (Proteintech, Chicago, IL, USA; 66009-1-Ig; Dilution multiple 1:2000), Atg5 (Proteintech, Chicago, IL, USA; 10181-2-AP; Dilution multiple 1:1000), Atg7 (Proteintech, Chicago, IL, USA; 67341-1-Ig; Dilution multiple 1:1000), Atg12 (Abclonal, Wuhan, China; A22788; Dilution multiple 1:1000), LC3 (Abclonal, Wuhan, China; A12319; Dilution multiple 1:2500), and p62 (Proteintech, Chicago, IL, USA; 66184-1-Ig; Dilution multiple 1:2000) were utilized for the Western blot, along with HRP-conjugated Goat Anti-Rabbit/Mouse IgG(H + L) (Proteintech, Chicago, IL, USA; SA00001-2/-1; 1:5000 dilution).

Techniques: Infection, Expressing, Western Blot, Control, Transfection, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: LncRNA NR_003508 Boosts Pseudomonas aeruginosa -Induced Autophagy by Facilitating the Conversion of LC3-I to LC3-II and p62 Degradation

doi: 10.3390/cells15141306

Figure Lengend Snippet: LncRNA NR_003508 promotes the relevant factors expression of autophagosome formation with PA infection. ( A ) Western blot was used to detect the expression of autophagy proteins in mouse lung tissue by intratracheal instillation of PA and si- NR_003508 . ( B ) The gray value analysis results. * p < 0.05 vs. NC, * p < 0.05, ** p < 0.01 vs. NC and PA. ( C ) The mRNA levels of autophagy-related factors were detected by q-PCR in mouse lung tissue by si- NR_003508 transfection. * p < 0.05, ** p < 0.01 vs. NC and PA. ( D ) IHC was used to detect the levels of autophagy proteins in mouse lung tissue with si- NR_003508 transfection (400×, scale bar = 100 μm). The tissue section structure is intact. Compared with the control group, there is lymphocyte infiltration in the interstitium of the PA infection group, and the cells are significantly recruited (the cell nuclei are blue), while the number of antibody-positive cells significantly increases (yellow to brown) and the positive cells significantly decrease in the si- NR_003508 + PA group. The expression of Atg5, Atg7, Atg12, LC3, and p62 in RAW264.7 ( E ) and BMDM ( H ) was detected by Western blot with LncRNA NR_003508 knockdown. The gray scale analysis results in RAW264.7 ( F ) and BMDM ( I ). * p < 0.05 vs. NC and PA, ** p < 0.01 vs. NC and PA. q-PCR was used to detect the expression of Atg5, Atg7, Atg12, and p62 in RAW264.7 ( G ) and BMDM ( J ) with LncRNA NR_003508 knockdown. * p < 0.05 vs. PA, ** p < 0.01 vs. NC and PA, *** p < 0.001 vs. NC and PA. Three independent experiments were performed as mean ± SD ( n = 3, each group).

Article Snippet: The primary antibodies β-actin (Proteintech, Chicago, IL, USA; 66009-1-Ig; Dilution multiple 1:2000), Atg5 (Proteintech, Chicago, IL, USA; 10181-2-AP; Dilution multiple 1:1000), Atg7 (Proteintech, Chicago, IL, USA; 67341-1-Ig; Dilution multiple 1:1000), Atg12 (Abclonal, Wuhan, China; A22788; Dilution multiple 1:1000), LC3 (Abclonal, Wuhan, China; A12319; Dilution multiple 1:2500), and p62 (Proteintech, Chicago, IL, USA; 66184-1-Ig; Dilution multiple 1:2000) were utilized for the Western blot, along with HRP-conjugated Goat Anti-Rabbit/Mouse IgG(H + L) (Proteintech, Chicago, IL, USA; SA00001-2/-1; 1:5000 dilution).

Techniques: Expressing, Infection, Western Blot, Transfection, Control, Knockdown

Journal: Cells

Article Title: LncRNA NR_003508 Boosts Pseudomonas aeruginosa -Induced Autophagy by Facilitating the Conversion of LC3-I to LC3-II and p62 Degradation

doi: 10.3390/cells15141306

Figure Lengend Snippet: LncRNA NR_003508 boosts autophagosome formation. ( A ) The expression of LC3 and p62 in RAW264.7 was detected by Western blot transfecting with si- NR_003508 , RAPA, and 3-MA. ( B ) The gray scale analysis results. * p < 0.05, ** p < 0.01 vs. si- NR_003508 . ( C ) The changes in the tandem mRFP-GFP-LC3 punctas were observed by confocal microscope (scale = 2.5 μm). ( D ) Quantitative analysis of yellow and red fluorescent spots. * p < 0.05, ** p < 0.01 vs. si- NR_003508 . Results of three independent experiments were shown as mean ± SD ( n = 3, each group).

Article Snippet: The primary antibodies β-actin (Proteintech, Chicago, IL, USA; 66009-1-Ig; Dilution multiple 1:2000), Atg5 (Proteintech, Chicago, IL, USA; 10181-2-AP; Dilution multiple 1:1000), Atg7 (Proteintech, Chicago, IL, USA; 67341-1-Ig; Dilution multiple 1:1000), Atg12 (Abclonal, Wuhan, China; A22788; Dilution multiple 1:1000), LC3 (Abclonal, Wuhan, China; A12319; Dilution multiple 1:2500), and p62 (Proteintech, Chicago, IL, USA; 66184-1-Ig; Dilution multiple 1:2000) were utilized for the Western blot, along with HRP-conjugated Goat Anti-Rabbit/Mouse IgG(H + L) (Proteintech, Chicago, IL, USA; SA00001-2/-1; 1:5000 dilution).

Techniques: Expressing, Western Blot, Microscopy

Journal: Cells

Article Title: LncRNA NR_003508 Boosts Pseudomonas aeruginosa -Induced Autophagy by Facilitating the Conversion of LC3-I to LC3-II and p62 Degradation

doi: 10.3390/cells15141306

Figure Lengend Snippet: LncRNA NR_003508 targets autophagy-related miRNA. ( A ) Prediction indicator of the interaction between LncRNA NR_003508 and autophagy proteins LC3 and p62-related miRNA (left are associated with LC3, right are associated with p62). The expression of LC3 ( B ) and p62 ( C ) miRNA was detected by q-PCR in different MOIs of PA infection. * p < 0.05 vs. Control. q-PCR was used to detect the expression of LC3 ( D ) and p62 ( E ) miRNA with LncRNA NR_003508 interference. ** p < 0.01 vs. NC. The LC3 ( F ) and p62 ( G ) expression with PA infection and LncRNA NR_003508 interference was detected by q-PCR. * p < 0.05 vs. NC and PA, ** p < 0.01 vs. PA. Three independent experiments were shown as mean ± SD ( n = 3).

Article Snippet: The primary antibodies β-actin (Proteintech, Chicago, IL, USA; 66009-1-Ig; Dilution multiple 1:2000), Atg5 (Proteintech, Chicago, IL, USA; 10181-2-AP; Dilution multiple 1:1000), Atg7 (Proteintech, Chicago, IL, USA; 67341-1-Ig; Dilution multiple 1:1000), Atg12 (Abclonal, Wuhan, China; A22788; Dilution multiple 1:1000), LC3 (Abclonal, Wuhan, China; A12319; Dilution multiple 1:2500), and p62 (Proteintech, Chicago, IL, USA; 66184-1-Ig; Dilution multiple 1:2000) were utilized for the Western blot, along with HRP-conjugated Goat Anti-Rabbit/Mouse IgG(H + L) (Proteintech, Chicago, IL, USA; SA00001-2/-1; 1:5000 dilution).

Techniques: Expressing, Infection, Control

Journal: Cells

Article Title: LncRNA NR_003508 Boosts Pseudomonas aeruginosa -Induced Autophagy by Facilitating the Conversion of LC3-I to LC3-II and p62 Degradation

doi: 10.3390/cells15141306

Figure Lengend Snippet: LncRNA NR_003508 sponges miR-344i to target LC3 and stimulates the conversion of LC3I to LC3II. ( A , B ) q-PCR was used to detect miR-344i levels at different times and MOIs of PA infection. ** p < 0.01 vs. Control. ( C ) Bielefeld Bioinformatics Service identified the binding sites for LncRNA NR_003508 and miR-344i. ( D ) The luciferase reporter assay confirmed the interaction between LncRNA NR_003508 and miR-344i ( n = 3). ** p < 0.01 vs. the NC group. ( E ) RIP verified the connection between LncRNA NR_003508 and miR-344i. ** p < 0.01, *** p < 0.001 vs. the IgG group. ( F ) RNA22 V2 prediction software was used for bioinformatics analysis. ( G ) The interaction between miR-344i and LC3 was validated by a dual-luciferase reporter assay. ** p < 0.01 vs. the NC group. ( H ) RIP experiment verified the relationship between miR-344i and LC3. ** p < 0.01, *** p < 0.001 vs. the IgG group. ( I ) Immunofluorescence was utilized to observe the co-localization of LncRNA NR_003508 , miR-344i, and LC3. (scale bar = 7.5 μm). ( J ) The conversion of LC3I to LC3II with miR-344i mimic transfection was detected by Western blot. ( K ) The gray value analysis results. ** p < 0.01 vs. the NC and PA group. The results are presented as mean ± SD from three separate experiments ( n = 3, each group).

Article Snippet: The primary antibodies β-actin (Proteintech, Chicago, IL, USA; 66009-1-Ig; Dilution multiple 1:2000), Atg5 (Proteintech, Chicago, IL, USA; 10181-2-AP; Dilution multiple 1:1000), Atg7 (Proteintech, Chicago, IL, USA; 67341-1-Ig; Dilution multiple 1:1000), Atg12 (Abclonal, Wuhan, China; A22788; Dilution multiple 1:1000), LC3 (Abclonal, Wuhan, China; A12319; Dilution multiple 1:2500), and p62 (Proteintech, Chicago, IL, USA; 66184-1-Ig; Dilution multiple 1:2000) were utilized for the Western blot, along with HRP-conjugated Goat Anti-Rabbit/Mouse IgG(H + L) (Proteintech, Chicago, IL, USA; SA00001-2/-1; 1:5000 dilution).

Techniques: Infection, Control, Binding Assay, Luciferase, Reporter Assay, Software, Immunofluorescence, Transfection, Western Blot

Journal: Metabolites

Article Title: Regulation of Mitophagy by Low-Intensity Pulsed Ultrasound Attenuates Endothelial Dysfunction

doi: 10.3390/metabo16050329

Figure Lengend Snippet: LIPUS promotes mitophagy in high glucose-stimulated HUVECs. ( A – D ) Representative protein bands and quantitative analysis of p62, LC3-II, and TOM20 protein levels; one-way ANOVA ( n = 3). ( E ) Immunofluorescence co-staining of LC3 (green), TOM20 (red), and DAPI (blue). The scale bar was set to 20 μm ( n = 3). ( F ) 3D surface plot of LC3 fluorescence. ( G ) Fluorescence intensity distribution profiles for LC3 and TOM20. Green represents LC3; red represents TOM20. ( H ) Quantification of LC3-TOM20 co-localization. Co-localization was quantified with the overlap coefficient calculated with ImageJ (Colocalization Finder plugin). A total of 16–21 cells per group were analyzed; one-way ANOVA ( n = 3); ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent SD of the mean.

Article Snippet: The primary antibody, Rabbit anti-LC3 antibody (1:200, Abmart, T55992 ) and mouse anti-TOM20 antibody (1:200, Proteintech, Wuhan, China, 666777-1-Ig), diluted in blocking solution, was applied overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: ANXA3 did not regulate myocardial free fatty acid uptake or lipolysis but positively modulated macroautophagy in T2DM mice. (A-C) Relative p62 protein expression level and LC3 II/I ratio in heart tissues of db/m and db/db mice analyzed by Western blot, n = 4. (D-F) Relative p62 protein expression level and LC3 II/I ratio in heart tissues of mice analyzed by Western blot, n = 4. (G-I) Relative p62 protein expression level and LC3 II/I ratio in AC16 cardiomyocytes analyzed by Western blot, n = 6. (J-L) Relative p62 protein expression level and LC3 II/I ratio in AC16 cardiomyocytes analyzed by Western blot, n = 6-8. The data were presented as the mean ± SD. ∗ P < 0.05 versus the db/m group or the db/db + OE-GFP group; ∗∗ P < 0.01 versus the db/m group or the vehicle group or the OE-NC + vehicle group; ∗∗∗ P < 0.001 versus the vehicle group or the OE-NC + vehicle group; # P < 0.05 versus the OE-NC + HGPA group; ### P < 0.001 versus the OE-NC + HGPA group.

Article Snippet: Blocked with 5% (w/v) non-fat-dried milkat room temperature for 1 h. Then the membranes were incubated with the anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:1000 [v/v]), the anti-YY1 antibody (Proteintech, 22156-1-AP; 1:1000 [v/v]) the anti-PLIN2 antibody (Proteintech, 15294-1-AP; 1:1000 [v/v]), the anti-SQSTM1/p62 antibody (Cell Signaling Technology, 23214; 1:1000 [v/v]), the anti-LC3 antibody (Cell Signaling Technology, 12741; 1:1000 [v/v]), the anti-Rab7 antibody (Proteintech, 55469-1-AP; 1:1000 [v/v])or the rabbit anti-α-Tubulin antibody (ABclonal, AC031; 1:1000 [v/v])overnight at 4 °C.

Techniques: Expressing, Western Blot