phospho sqstm1 p62  (Cell Signaling Technology Inc)

Journal: Frontiers in Pharmacology

Article Title: Baicalin chemosensitivity enhancement of cisplatin in bladder cancer via autophagy flux inhibition

doi: 10.3389/fphar.2026.1676788

Figure Lengend Snippet: Cisplatin induces autophagy in BC T24 and BIU-87 cells. (A,B) T24 and BIU-87 cells were analyed by western blotting using anti-LC3B and anti-p62 antibodies after treating with cisplatin (1 μg/ml) for 24 h (C-E) T24 and BIU-87 cells were treated with cisplatin (1 μg/ml) for 24 h before being labeled with immunofluorescence staining of LC3B (red). The nuclei were counterstained with DAPI (blue). Scale bar: 20 µm. (F) The subcellular structure of BC T24 and BIU-87 cells after treatment with or without cisplatin (1 μg/ml) for 24 h were imaged by TEM (The green triangle are autophagosome, while the red triangle are autolysosomes). Scale bar: 1 μm. All the data were presented as means ± S.D. and are representative of three independent experiments. **P < 0.01; ***P < 0.001.

Article Snippet: Then the sections were incubated with 0.3% H 2 O 2 for 10 min at room temperature, washed with PBS and blocked in 5% BSA at 37 °C for 30 min, followed by incubated with primary antibodies: LAMP1 (1:200, 21997, Proteintech), CTSD (1:500, ab75852, Abcam), LC3B (1:200, 83506, CST), Phospho-SQSTM1/p62 (1:200, 16177, CST) overnight at 4 °C and the secondary antibody for 1 h at room temperature.

Techniques: Western Blot, Labeling, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Baicalin chemosensitivity enhancement of cisplatin in bladder cancer via autophagy flux inhibition

doi: 10.3389/fphar.2026.1676788

Figure Lengend Snippet: Confocal images of BC T24 and BIU-87 cells infected with adenovirus mCherry-GFP-LC3B (mCherry: red, eGFP: green, Hoechst: bule). (A-C) Western blot analysis of the protein level of LC3B I, LC3B II and P62 in T24 and BIU-87 cells treated with DMSO or baicalin (40 μM, 80 μM) for 24 h (D,E) The relative mRNA expression levels of LAMP1, LAMP2, CTSB, and CTSD were detected in T24 cells and BIU-87 cells following treatment with DMSO (as a control) or baicalin (40 μM). (F-I) The cells were infected with adenovirus for 24 h and then incubated with DMSO, cisplatin (1 μg/mL), Rapa (2.5 μM), CQ (25 μM), and baicalin (40 μM) for 24 h. Nuclei were stained with Hoechst. Scale bar: 20 μm. All the data were presented as means ± S.D. and are representative of three independent experiments. **P < 0.01; ***P < 0.001.

Article Snippet: Then the sections were incubated with 0.3% H 2 O 2 for 10 min at room temperature, washed with PBS and blocked in 5% BSA at 37 °C for 30 min, followed by incubated with primary antibodies: LAMP1 (1:200, 21997, Proteintech), CTSD (1:500, ab75852, Abcam), LC3B (1:200, 83506, CST), Phospho-SQSTM1/p62 (1:200, 16177, CST) overnight at 4 °C and the secondary antibody for 1 h at room temperature.

Techniques: Infection, Western Blot, Expressing, Control, Incubation, Staining

Journal: Frontiers in Pharmacology

Article Title: Baicalin chemosensitivity enhancement of cisplatin in bladder cancer via autophagy flux inhibition

doi: 10.3389/fphar.2026.1676788

Figure Lengend Snippet: Baicalin blocked the autophagic flux of BC in vivo by inhibiting lysosome activity. (A-C) The expression of LC3B, p62, CTSB, CTSD, LAMP1, and LAMP2 of xenograft tumors was detected by western blotting. (D,E) Immunohistochemical staining was performed on tumor tissue using LC3B, P62, LAMP1, CTSB, and CTSD antibodies. Scale bar: 100 μm. All the data were presented as means ± S.D. and are representative of three independent experiments. *P < 0.05 vs . PBS ; **P < 0.01 vs . PBS ; ***P < 0.001 vs . PBS.

Article Snippet: Then the sections were incubated with 0.3% H 2 O 2 for 10 min at room temperature, washed with PBS and blocked in 5% BSA at 37 °C for 30 min, followed by incubated with primary antibodies: LAMP1 (1:200, 21997, Proteintech), CTSD (1:500, ab75852, Abcam), LC3B (1:200, 83506, CST), Phospho-SQSTM1/p62 (1:200, 16177, CST) overnight at 4 °C and the secondary antibody for 1 h at room temperature.

Techniques: In Vivo, Activity Assay, Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of the Tau aggregation system. B) Confocal images of Tau-mRuby expressing cells treated with and without in vitro formed Tau fibrils (scale bar = 20 µm). C) Immunofluorescence images of Tau, p62 and ubiquitin in cells upon treatment with Tau fibrils (scale bar = 20 µm). D) Confocal images of Tau in WT, 5KO and 5KO cell lines re-expressing p62 (scale bar = 20 µm). E) Quantification of number of Tau aggregates per cell in D. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the average number of aggregates of a single image. One-way ANOVA with Dunnett’s multiple comparisons test. F) Confocal images of 5KO Tau-mRuby cells re-expressing mClover-p62, mClover-p62ΔPB1 or mClover-p62ΔUBA (scale bar = 20 µm). G) Pearson’s correlation (PCC) between Tau aggregates and mClover-p62, mClover-p62ΔPB1 and mClover-p62ΔUBA in F. The data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the measured correlation within an individual cell. One-way ANOVA with Dunnett’s multiple comparisons test. H) Quantification of the number of Tau aggregates per cell in F. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the average number of aggregates of a single image. One-way ANOVA with Dunnett’s multiple comparisons test. I) Immunofluorescence images of p62 in cells stably expressing Tau-mRuby untreated or treated with Tau fibrils to induce Tau aggregation in the cells (scale bar = 20 µm). J) Quantification of the number of p62 puncta per image taken in I. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the average number of aggregates in a single image. Unpaired t-test. K) Representative fluorescence-activated cell sorting (FACS) plots of Tau flux measurements upon aggregation. L) Quantification of Tau flux upon Tau aggregation in C. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). One-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05, ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and their dilutions were used in this study: rabbit anti-Tau (1:500; MA5-4245, Invitrogen), mouse anti-p62 (1:100; #610832, Clone 3/p62 lck ligand, BD Bioscience), rabbit anti-TAX1BP1 (1:100; #5105, clone D1D5, CST), rabbit anti-p62 p-S403 (1:100; #39786, clone D8D67, CST), rabbit anti-FIP200 (1:200; #12436, clone D10D11, CST), rabbit anti-TBK1 p-S172 (1:100; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:100; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:100; #8054, clone D8H5, CST), mouse anti-LC3B (1:100; 0260-100/LC3-2G6, NanoTools), mouse anti-ubiquitin (UBCJ2 (1:500; ENZ-ABS840, Enzo Life Sciences).

Techniques: Expressing, In Vitro, Immunofluorescence, Ubiquitin Proteomics, Stable Transfection, Fluorescence, FACS

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of the pelleting assay. B) Immunoblotting for autophagy factors after Tau aggregation and the pelleting assay. C) Quantification of the signal in the pellet fraction in comparison to the input samples. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Multiple unpaired t-tests. D) Immunofluorescence images of Tau, p62, TAX1BP1, ATG13 and TBK1 p-S172 (scale bar = 20 µm). E) Pearson’s correlation (PCC) between Tau aggregates and p62, TAX1BP1, ATG13 and TBK1 p-S172. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the measured correlation within an individual cell. One-way ANOVA with Dunnett’s multiple comparisons test. F) Immunofluorescence images for p62 and p62 p-S403 in cells expressing Tau-mRuby after Tau seeding (scale bar = 20 µm). G) Pearson’s correlation (PCC) between Tau aggregates and p62 or p62 p-S403 in I. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to measured correlation within an individual cell. H) Schematic summary of the results. *P < 0.05, **P < 0.005, ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and their dilutions were used in this study: rabbit anti-Tau (1:500; MA5-4245, Invitrogen), mouse anti-p62 (1:100; #610832, Clone 3/p62 lck ligand, BD Bioscience), rabbit anti-TAX1BP1 (1:100; #5105, clone D1D5, CST), rabbit anti-p62 p-S403 (1:100; #39786, clone D8D67, CST), rabbit anti-FIP200 (1:200; #12436, clone D10D11, CST), rabbit anti-TBK1 p-S172 (1:100; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:100; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:100; #8054, clone D8H5, CST), mouse anti-LC3B (1:100; 0260-100/LC3-2G6, NanoTools), mouse anti-ubiquitin (UBCJ2 (1:500; ENZ-ABS840, Enzo Life Sciences).

Techniques: Western Blot, Comparison, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Immunoblotting of phospho-ULK1, ATG13, TBK1 and p62 upon Tau aggregation and TAX1BP1 tethering. B) Quantification of relative increase of phosphorylation levels upon TAX1BP1 tethering. Phosphorylation signal was normalized to the total protein. To account for inter-experimental variability, values are expressed as a relative to the -Rapalog samples within each biological replicate. The data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.005. ns, not significant.

Article Snippet: The following primary antibodies and their dilutions were used in this study: rabbit anti-Tau (1:500; MA5-4245, Invitrogen), mouse anti-p62 (1:100; #610832, Clone 3/p62 lck ligand, BD Bioscience), rabbit anti-TAX1BP1 (1:100; #5105, clone D1D5, CST), rabbit anti-p62 p-S403 (1:100; #39786, clone D8D67, CST), rabbit anti-FIP200 (1:200; #12436, clone D10D11, CST), rabbit anti-TBK1 p-S172 (1:100; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:100; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:100; #8054, clone D8H5, CST), mouse anti-LC3B (1:100; 0260-100/LC3-2G6, NanoTools), mouse anti-ubiquitin (UBCJ2 (1:500; ENZ-ABS840, Enzo Life Sciences).

Techniques: Western Blot, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Rhein protects against renal aging and fibrotic injury by multiple targets through inhibition of TNF-α-mediated autophagy and necroptosis crosstalk

doi: 10.3389/fphar.2026.1693000

Figure Lengend Snippet: Rhein reduced autophagy through mTOR signaling in NRK-52E cells treated with D-gal. (A) WB analysis of p-mTOR, mTOR, p62, p-p62, beclin1, and LC3 in cultured NRK-52E cells exposed to 100 mM D-gal with or without rhein (1 and 3 μg/mL) and VE (50 μg/mL) treatment for 24 h (B) NRK-52E cells were transfected with pmRFP-LC3, treated with D-gal (100 mM) with or without rhein (3 μg/mL) and VE (50 μg/mL) treatment for 24 h, after which fluorescence microscopy was performed. Scale bar: 5 μm. (C) WB analysis of p-mTOR, mTOR, p-p62, p62, beclin1, and LC3 in cultured NRK-52E cells treated with 100 mM D-gal and rhein (3 μg/mL), rapamycin (100 μM), and MHY1485 (2 μM) for 24 h. (D) NRK-52E cells were transfected with pmRFP-LC3; treated with D-gal (100 mM) along with rhein (3 μg/mL), rapamycin (100 μM), and MHY1485 (2 μM) for 24 h, and then analyzed using fluorescence microscopy. Scale bar: 5 μm. Results are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01. Abbreviations: D-gal, D-galactose; p-mTOR, phosphorylated mTOR; p-p62, phosphorylated p62; WB, Western blot.

Article Snippet: The levels of the phosphorylated and total proteins of mTOR and the phosphorylated and total proteins of p62, beclin1, LC3, RIPK1, RIPK3, and caspase-8 were assessed using anti-phospho mTOR (Ser2448; 1:1000, #5536), anti-mTOR (1:1000, #2983), anti-phospho p62 (1:1000, #39786), anti-p62 (1:1000, #23214), anti-beclin1 (1:1000, #3495), anti-LC3A/B (1:1000, #12741), anti-RIPK1 (1:1000, #3493), anti-RIPK3 (1:1000, #15828), anti-caspase-8 (1:1000, #4790) and anti-cleaved-caspase-8 (1:1000, #8592) antibodies, respectively (Cell Signaling, Beverly, MA, United States).

Techniques: Cell Culture, Transfection, Fluorescence, Microscopy, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Rhein protects against renal aging and fibrotic injury by multiple targets through inhibition of TNF-α-mediated autophagy and necroptosis crosstalk

doi: 10.3389/fphar.2026.1693000

Figure Lengend Snippet: Rhein alleviated D-gal-induced renal aging and fibrotic injury by TNF-α-mediated necroptosis and autophagy in rats. (A) Measurement of serum TNF-α level of rats in the Normal, Model, Rhein-low, Rhein-high, and VE groups. (B) WB analysis of RIPK1, RIPK3, cleaved-caspase-8, and p-MLKL in kidneys in the Normal, Model, Rhein-low, Rhein-high, and VE groups. (C) WB analysis of p-mTOR, mTOR, p-p62, p62, beclin1, and LC3 in kidneys in the Normal, Model, Rhein-low, Rhein-high, and VE groups. (D) Typical images of immunohistochemical staining of LC3 in kidneys in the Normal, Model, Rhein-low, Rhein-high, and VE groups. Scale bar: 20 μm. (E) The quantity changes of autophagosome in kidneys in the Normal, Model, Rhein-low, Rhein-high, and VE groups by transmission electron microscopy. Scale bar: 1 μm. The black arrows indicate autophagosomes, which are distinctive for their double-membrane structure. (F) WB analysis of p-mTOR, mTOR, p-p62, p62, beclin1, and LC3 in kidneys in the Normal, Model, Rhein-low, Rapamycin, and MHY1485 groups. (G) WB analysis of klotho, p53, and p21 in kidneys in the Normal, Model, Rhein-low, Rapamycin, and MHY1485 groups. Results are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01. Abbreviations: D-gal, D-galactose; TNFR1, tumor necrosis factor receptor-1; p-mTOR, phosphorylated mTOR; p-p62, phosphorylated p62; WB, Western blot.

Article Snippet: The levels of the phosphorylated and total proteins of mTOR and the phosphorylated and total proteins of p62, beclin1, LC3, RIPK1, RIPK3, and caspase-8 were assessed using anti-phospho mTOR (Ser2448; 1:1000, #5536), anti-mTOR (1:1000, #2983), anti-phospho p62 (1:1000, #39786), anti-p62 (1:1000, #23214), anti-beclin1 (1:1000, #3495), anti-LC3A/B (1:1000, #12741), anti-RIPK1 (1:1000, #3493), anti-RIPK3 (1:1000, #15828), anti-caspase-8 (1:1000, #4790) and anti-cleaved-caspase-8 (1:1000, #8592) antibodies, respectively (Cell Signaling, Beverly, MA, United States).

Techniques: Immunohistochemical staining, Staining, Transmission Assay, Electron Microscopy, Membrane, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Cancer-associated fibroblast-derived extracellular vesicles regulate lipophagy through PLIN2 to modulate dormancy in salivary gland adenoid cystic carcinoma cells

doi: 10.1038/s12276-025-01600-3

Figure Lengend Snippet: a , b Representative Nile Red staining images of dormant SACC cells overexpressing PLIN2 in the absence or presence of HCQ treatment. Scale bar, 20 μm. c qRT–PCR analysis of dormant SACC cells transfected with a NC or PLIN2-overexpressing vector to measure ER stress-related gene expression. d Western blot analysis of p-EIF2S1, EIF2S1, p-PERK and PERK levels in dormant SACC cells. e The dormant SACC cells were incubated with PERK inhibitor GSK2606414 (10 μM). Then, the protein levels were determined by western blot. f Molecular modeling analysis of the interaction between PLIN2 and the binding site within the p62 domain. g , h Co-IP of p62, followed by western blot for p62 and PLIN2, in dormant SACC-LM cells ( g ) and SACC-LM cells overexpressing PLIN2 ( h ).

Article Snippet: The following antibodies were used: anti-NR2F1 (ab181137) and anti-LC3 (ab192890) from Abcam; anti-Atg5 (12994), anti-p-mTOR (5536) and anti-p62 (16177) from Cell Signaling Technology; anti-caspase-3 (68773-1-Ig), anti-p-ATK (66444-1-Ig), anti-BAX (50599-2-Ig), anti-FAP (11779-1-AP), anti-BCL2 (12789-1-AP) and anti-α-SMA (14395-1-AP) from Proteintech; anti-HSP70 (ET1601-11), anti-GAPDH (ET1601-4), anti-CD63 (ET1607-2), anti-p21 (HA722685), anti-PLIN2 (ET1704-17), anti-p27 (ET1608-61) and anti-EIF2S1 ( HA500385 ) from HUABIO; anti-p-PERK (340846), anti-PERK ( R25311 ) from Zen-Bio.

Techniques: Staining, Quantitative RT-PCR, Transfection, Plasmid Preparation, Gene Expression, Western Blot, Incubation, Binding Assay, Co-Immunoprecipitation Assay