Journal: Cell Death Discovery

Article Title: Targeting ESR1 restores SQSTM1-dependent autophagy and sensitizes ER-positive breast cancer to oxidative and radiation stress

doi: 10.1038/s41420-025-02755-8

Figure Lengend Snippet: A Protein levels of Bcl-2, caspase 9 (full length/cleaved) and caspase 3 in T47D with or without ESR1 knockdown in a radiation dose-dependent manner. Actin was used as internal control. B Protein levels of COX2 and GPX4 in T47D with or without ESR1 knockdown in a radiation dose-dependent manner. Actin was used as internal control. C Protein levels of p-IRE1-α and IRE1-α in T47D with or without ESR1 knockdown in a radiation dose-dependent manner. Actin was used as internal control. D Protein levels of LC3B (I/II) and p62 in T47D isogenic model with or without CQ treatment. Actin was used as internal control. E Protein levels of LC3B (I/II) and p62 in T47D with or without ESR1 knockdown in a radiation dose-dependent manner. Actin was used as internal control. F Images of LC3B in ESR1 overexpression or knockdown model by immunofluorescence, respectively. DAPI was used as internal control. G Images of LAMP2 in ESR1 overexpression model by immunofluorescence. DAPI was used as internal control. The data from three independent experiments are presented as the means ± SEM.

Article Snippet: Phospho-p62 (Ser349) , 95697 , Cell signaling , 1:1000.

Techniques: Knockdown, Control, Over Expression, Immunofluorescence

Journal: Cell Death Discovery

Article Title: Targeting ESR1 restores SQSTM1-dependent autophagy and sensitizes ER-positive breast cancer to oxidative and radiation stress

doi: 10.1038/s41420-025-02755-8

Figure Lengend Snippet: A Protein levels of ATM, p-ATM (S1981), Ku70, Ku80, XRCC4, Rad51 and BRCA1 in T47D with or without ESR1 knockdown by H2O2 treatment. Actin was used as an internal control. B Images of TP53BP1 and γ-H2AX in T47D ESR1 knockdown model by H 2 O 2 treatment by immunofluorescence. Hoechst was used as an internal control. C Protein levels of ATM, p-ATM (S1981), Ku70, Ku80, XRCC4 and Rad51 in MCF7 with or without ESR1 overexpression by H 2 O 2 treatment. Actin was used as an internal control. D Images of TP53BP1 and γ-H2AX in MCF7 ESR1 overexpression model by H 2 O 2 treatment via immunofluorescence. Hoechst was used as an internal control. E Protein levels of LC3B (I/II) and p62 in T47D ESR1 knockdown model with or without CQ/ H 2 O 2 . Actin was used as an internal control. F Images of LC3B in ESR1 overexpression and knockdown model by H 2 O 2 treatment via immunofluorescence. Hoechst was used as an internal control. G Protein levels of LC3B (I/II) and p62 in T47D ESR1 knockdown model treated with H 2 O 2 with or without 3-MA. Actin was used as an internal control. H Images of LC3B in the MCF7 ESR1 overexpression model were taken after treatment with H₂O₂ for one hour and with or without 3-MA for three hours. Hoechst was used as the internal control. The data from three independent experiments are presented as the means ± SEM.

Article Snippet: Phospho-p62 (Ser349) , 95697 , Cell signaling , 1:1000.

Techniques: Knockdown, Control, Immunofluorescence, Over Expression

Journal: Cell Death Discovery

Article Title: Targeting ESR1 restores SQSTM1-dependent autophagy and sensitizes ER-positive breast cancer to oxidative and radiation stress

doi: 10.1038/s41420-025-02755-8

Figure Lengend Snippet: A Electrophoretic separation and Coomassie blue staining of MCF7 with and without ESR1 overexpression. B Venn diagram showing the overlapping molecules between ESR1 IP, vector IP and ESR1 with MG-132 treatment. C Top ranking of selected molecules from ESR1-based proteomics profiles. D Interaction affinity between p62 and ESR1 by pull-down assay in ESR1 overexpression model. E Protein levels of p62 in MCF7 ESR1 overexpression by MG-132 treatment. Actin was used as internal control. F Protein levels of phosphor-p62, p62 and ESR1 in MCF7 cells with and without E2 treatment. Actin was used as internal control. G Interaction affinity between p62 and ESR1 by pull-down assay after E2 treatment. H Survival fraction of ESR1 knockdown model with or without E2 treatment followed by irradiation. ( I ) Protein levels of LC3B and p62 in T-47D ESR1 isogenic model. Actin was used as internal control. ( J ) Images of TP53BP1 and γ-H2AX in T47D ESR1 knockdown model by E2 treatment combined with H2O2 by immunofluorescence. Hoechst was used as internal control. K Images of LC3B in T47D ESR1 knockdown model by E2 treatment, E2 combined with p62 overexpression and TamR via immunofluorescence. Hoechst was used as internal control. The data from three independent experiments are presented as the means ± SEM.

Article Snippet: Phospho-p62 (Ser349) , 95697 , Cell signaling , 1:1000.

Techniques: Staining, Over Expression, Plasmid Preparation, Pull Down Assay, Control, Knockdown, Irradiation, Immunofluorescence

Journal: bioRxiv

Article Title: Autophagy-Dependent Regulation of YAP1 by STK38 Governs Recruitment of Differentiated Cells as Progenitor Cells During Regeneration

doi: 10.1101/2025.04.14.648819

Figure Lengend Snippet: (A) Western blot showing active-YAP1, p62, p-p62T269/272, p-p62-S351, p-p62-S405, and β-actin loading control in whole corpus protein extracts from C57BL/6J mice at various injury time points. (B) Confocal, z-stack immunofluorescence showing STK38 is often found within LC3 vesicles in chief cells in the early stage of paligenosis. Scale bar, 5 µm (C,D) Immunoprecipitation assays demonstrate that overexpressed, myc- and mCherry-GFP-tagged STK38 and LC3 can be co-immunoprecipitated in 293-T cells. “Input” = Total protein lysates of 293-T cells co-transfected with empty vector and mCherry-GFP-LC3 plasmid, or myc-STK38 and mCherry-GFP-LC3 plasmids(C), or empty vector and myc-STK38 plasmid, or myc-STK38 and mCherry-GFP-LC3 plasmids(D). “S.E.” and “L.E.” = short and long exposure. Asterisk denotes molecular mass of tagged STK38, which can be distinguished from immunoglobulin heavy chain seen in input. (E) Western blot demonstrates that overexpression of UMF1 in AGS human gastric cell line is sufficient to protect STK38 from degradation induced by rapamycin treatment. (F) Pairwise gene expression correlation analysis using TCGA normal stomach dataset and GTEx stomach dataset reveals a moderate positive linear correlation between UFM1 and STK38 expression. (p=8.9e-16, R=0.52, generated by http://gepia.cancer-pku.cn/ ). (G,H) Western blot analysis shows that Rapamycin inhibits mTORC1 activity and reduces STK38 protein levels in human (G) AGS (gastric), (H) Hep3B2.1-7 (liver), and 293-T (kidney) cell lines. (I) Western blot shows that MG132 proteasome inhibitor not only does not lead to increased STK38 but results in decrease STK38 protein abundance in a dose-dependent manner in the AGS cell line. (J) IHC of wild-type C57BL/6J stomach tissue demonstrates that rapamycin treatment (60 μg/20 g body weight, euthanized 24hours post-injection) effectively reduces the expression of STK38 in the stomach. (K) Western blot demonstrates that rapamycin treatment effectively reduces STK38 protein expression in vivo , as evidenced by gastric protein lysates obtained from experiments like the one depicted in panel J. (L) IHC of wild-type C57BL/6J stomach tissue showing decreased UFM1 in chief cell paligenosis. Red brackets point out the location of the chief cell zone. Scale bar, 50 µm (M) IHC of wild-type C57BL/6J stomach tissue shows that Lys05 treatment effectively preserves STK38 protein levels in HDTAM-treated stomach tissue during stage 3. Scale bar, 100 µm. (N) Immunofluorescence showing reduced STK38 in chief cells (GIF+ cells) of Mist1 −/− mice compared to control. Scale bar, 10 µm (O) IHC of stomach tissue shows active YAP1 increase in chief cells of Mist1 −/− mice (right) chief cells compared to control (left). Red dashed lines point out the outline of chief cells. Note YAP1-positive parietal cells serve as positive controls for immunostaining. Scale bar, 10 µm.

Article Snippet: Membranes were incubated with 3% bovine serum albumin (BSA) overnight at 4 °C with various primary antibodies: anti-STK38 (1:1,000, Proteintech), anti-STK38L (1:1,000, Proteintech), anti-Phospho-STK38/STK38L (Thr444, Thr442, 1:1,000, Thermo Fisher), anti-active YAP1 (1:1,500, Abcam), anti-Phospho-YAP (Ser127, 1:1,000, CST), anti-Phospho-YAP (Ser397, 1:1,000, CST), anti-YAP (1:2,000, CST), anti-Phospho-SQSTM1/p62 (1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser403, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser349, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Thr269/Ser272, 1:1,000, CST), anti-LATS1 (1:1,000, CST), anti-Phospho-LATS1 (Ser909, 1:1,000, CST), anti-Phospho-LATS1 (Thr1079, 1:1,000, CST), anti-NF2 (1:1,000, CST), anti-MST1 (1:1,000, CST), anti-MST2 (1:1,000, CST), anti-Phospho-MST1 (Thr183)/MST2 (Thr180, 1:1,000, CST), anti-MOB1 (1:1,000, CST), anti-SAV1 (1:1,000, CST), anti-RASSF1 (1:1,000, Invitrogen), anti-LC3B (1:2,000, Novus Biologicals), anti-UFM1 (1:1,000, Abcam), anti-Phospho-S6 Ribosomal Protein (Ser240/244, 1:1,000, CST), and anti-beta Actin (1:1,000, Santa Cruz).

Techniques: Western Blot, Control, Immunofluorescence, Immunoprecipitation, Transfection, Plasmid Preparation, Over Expression, Gene Expression, Expressing, Generated, Activity Assay, Quantitative Proteomics, Injection, In Vivo, Immunostaining

Journal: bioRxiv

Article Title: Autophagy-Dependent Regulation of YAP1 by STK38 Governs Recruitment of Differentiated Cells as Progenitor Cells During Regeneration

doi: 10.1101/2025.04.14.648819

Figure Lengend Snippet: (A) Increased levels of active p62 are observed in the pancreas of Cerulein-treated C57BL/6J mice. (B) Total abundance of canonical Hippo pathway kinases remain unchanged in Atf3 −/− mice during paligenosis, and the decrease seen in STK38 early in paligenosis is less marked with STK38 also persisting throughout paligenosis. (C) Quantification of panel B highlights how STK38 abundance declined more gradually and less markedly in Atf3 −/− mice compared to wild-type mice. (D) IHC staining revealed a decrease in STK38 levels in the pancreas of rapamycin-treated mice. Scale bar, 100 µm. (E,F) IHC staining demonstrated a reduction of UMF1 in Mist1 knockout mouse gastric glands (E) and acinar cells (F). Scale bar, 50 µm. (G,H) IHC staining illustrated a decrease in STK38 levels in the stomach (G) and pancreas (H) of Mist1 knockout mice. Scale bar, 50 µm. (I,J) Immunofluorescence staining shows upregulation of active YAP1 in plasma cells of Mist1 knockout mice compared to controls. The immunofluorescence staining of plasma cells (I) with subsequent statistical analysis confirms finding by (J). Scale bar, 50 µm. **** P < 0.0001 (unpaired t-test). Data are presented as mean ± SEM, derived from 10 low-power fields per condition across three independent experiments.

Article Snippet: Membranes were incubated with 3% bovine serum albumin (BSA) overnight at 4 °C with various primary antibodies: anti-STK38 (1:1,000, Proteintech), anti-STK38L (1:1,000, Proteintech), anti-Phospho-STK38/STK38L (Thr444, Thr442, 1:1,000, Thermo Fisher), anti-active YAP1 (1:1,500, Abcam), anti-Phospho-YAP (Ser127, 1:1,000, CST), anti-Phospho-YAP (Ser397, 1:1,000, CST), anti-YAP (1:2,000, CST), anti-Phospho-SQSTM1/p62 (1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser403, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser349, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Thr269/Ser272, 1:1,000, CST), anti-LATS1 (1:1,000, CST), anti-Phospho-LATS1 (Ser909, 1:1,000, CST), anti-Phospho-LATS1 (Thr1079, 1:1,000, CST), anti-NF2 (1:1,000, CST), anti-MST1 (1:1,000, CST), anti-MST2 (1:1,000, CST), anti-Phospho-MST1 (Thr183)/MST2 (Thr180, 1:1,000, CST), anti-MOB1 (1:1,000, CST), anti-SAV1 (1:1,000, CST), anti-RASSF1 (1:1,000, Invitrogen), anti-LC3B (1:2,000, Novus Biologicals), anti-UFM1 (1:1,000, Abcam), anti-Phospho-S6 Ribosomal Protein (Ser240/244, 1:1,000, CST), and anti-beta Actin (1:1,000, Santa Cruz).

Techniques: Immunohistochemistry, Knock-Out, Immunofluorescence, Staining, Clinical Proteomics, Derivative Assay

Journal: Cells

Article Title: Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response.

doi: 10.3390/cells14030192

Figure Lengend Snippet: Figure 1. Chronic IL-1 sublines have basally high accumulation of the p62-KEAP1 interaction. (A) Mass spectrometry using gel LCMS was performed for p62 immunoprecipitates from LNCaP-1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum. Mass spectrometry was analyzed using Scaffold software. Values shown are Scaffold “exclusive spectrum count”. LNas1 and LNbs1 show high basal accumulation of the p62-KEAP1 interaction in 10% serum, whereas no KEAP1 interaction is detected in LNCaP-1 parentals. (B) p62 immunoprecipitation (IP) was performed for LNCaP-1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum, followed by western blot for p62 and KEAP1. LNas1 and LNbs1 show high basal accumulation of the p62-KEAP1 interaction. IgG IP is the negative control for p62 IP. ND = not detected.

Article Snippet: Primary antibodies: p62 (Abnova, Walnut, CA, USA; L2011-2C11), KEAP1 (Cell Signaling, Danvers, MA, USA; 8074T), p-p62 (Ser349) (Cell Signaling, Danvers, MA, USA; 95697), GCLC (Cell Signaling, Danvers, MA; 48005S), HO-1 (Cell Signaling, Danvers, MA, USA; 43966S), and β-actin (Santa Cruz, Santa Cruz, CA, USA; sc-69879).

Techniques: Mass Spectrometry, Software, Immunoprecipitation, Western Blot, Negative Control

Journal: Cells

Article Title: Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response.

doi: 10.3390/cells14030192

Figure Lengend Snippet: Figure 5. The p62-KEAP1 complex accumulation is upregulated by serum starvation in LNCaP parental cells and constitutive in subline cells. (A) DIA mass spectrometry was performed for LNCaP- 1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum or 0% serum for 2 days. p62 or KEAP1 were immunoprecipitated (IP) from LNCaP-1, LNas1, or LNbs1 cells. DIA mass spectrometry was analyzed using Scaffold DIA software. Values shown are non-normalized exclusive intensity. As compared to 10% serum, p62 and KEAP1 exclusive intensity are greater in 0% serum in LNCaP-1 parental cells for both p62 IP and KEAP1 IP, suggesting that 0% serum starvation induces accumulation of the p62-KEAP1 complex in LNCaP-1 cells. An increase in exclusive intensity in 0% versus 10% serum is not observed for LNas1 or LNbs1, suggesting that serum starvation does not induce p62-KEAP1 complex formation in the sublines. (B,C) IP followed by western blot was performed for LNCaP-1, LNas1, and LNbs1 cells grown in normal growth medium containing 10% serum or 0% serum for 2 days, and blots were probed for p62, p-p62 (SER349), or KEAP1. (B) p62 or (C) KEAP1 were IP’d from LNCaP-1, LNas1, or LNbs1 cells. Input western blots show that 0% serum induces p62 accumulation in LNCaP-1 parental cells, and IP western blots show that 0% induces p62-KEAP1 interaction in LNCaP-1 parental cells. Input and IP western blots show that in comparison to parental cells, p62 levels and p62-KEAP1 complex formation are basally higher in the sublines (e.g., 10% serum). In addition, input and IP western blots show that, in comparison to 10% serum, 0% serum reduces KEAP1 accumulation in the sublines, yet the p62-KEAP1 complex formation is comparable to parental 0% and subline 10%, suggesting the complex is constitutive and stable. Finally, p-p62 (SER349) is found only in the subline p62 immunoprecipitates. Thus, KEAP1 does not bind p-p62 (SER349) in the LNCaP-1 parental or subline cells grown in 10% or 0% serum. β-actin is the western blot loading control. IgG IP is the negative control.

Article Snippet: Primary antibodies: p62 (Abnova, Walnut, CA, USA; L2011-2C11), KEAP1 (Cell Signaling, Danvers, MA, USA; 8074T), p-p62 (Ser349) (Cell Signaling, Danvers, MA, USA; 95697), GCLC (Cell Signaling, Danvers, MA; 48005S), HO-1 (Cell Signaling, Danvers, MA, USA; 43966S), and β-actin (Santa Cruz, Santa Cruz, CA, USA; sc-69879).

Techniques: Mass Spectrometry, Immunoprecipitation, Software, Western Blot, Comparison, Control, Negative Control

Journal: Cells

Article Title: Chronic IL-1-Exposed LNCaP Cells Evolve High Basal p62-KEAP1 Complex Accumulation and NRF2/KEAP1-Dependent and -Independent Hypersensitive Nutrient Deprivation Response.

doi: 10.3390/cells14030192

Figure Lengend Snippet: Figure 8. Model. Chronic IL-1 exposure selects for cells that evolve high basal accumulation of NRF2-indepedent p62-KEAP1 interaction, the function of which remains unknown. Chronic IL-1 exposure also selects for cells that have high basal HMOX1 and GCLC and are hypersensitive to serum starvation-induced NRF2-KEAP1-dependent HMOX1 repression and NRF2-KEAP1-p62-independent GCLC upregulation. We speculate that under serum starvation conditions, hypersensitive HMOX1 repression and GCLC induction may protect the chronic IL-1 sublines from ferroptosis by reducing Fe2+ and increasing glutathione production, respectively.

Article Snippet: Primary antibodies: p62 (Abnova, Walnut, CA, USA; L2011-2C11), KEAP1 (Cell Signaling, Danvers, MA, USA; 8074T), p-p62 (Ser349) (Cell Signaling, Danvers, MA, USA; 95697), GCLC (Cell Signaling, Danvers, MA; 48005S), HO-1 (Cell Signaling, Danvers, MA, USA; 43966S), and β-actin (Santa Cruz, Santa Cruz, CA, USA; sc-69879).

Techniques:

Journal: Cell reports

Article Title: Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

doi: 10.1016/j.celrep.2024.115016

Figure Lengend Snippet: (A) Schematic of the VPS37A-containing ESCRT-I complex. UEV, ubiquitin-E2-like variant; UEVL, UEV-like; SOUBA, solenoid of overlapping UBAs. (B) Sequences of human and mouse VPS37A N termini. (C) Immunoblot analysis of VPS37A KO U-2 OS cells that were stably transduced with the indicated constructs and starved in the presence or absence of 100 nM bafilomycin A1 (BafA1) for 3 h. (D) Bar plots of LC3-II and p62 degradation ratios ([starvation + BafA1] / starvation) in (C) ( n = 3). (E) Confocal images of HaloTag (HT)-LC3-expressing U-2 OS cells that were starved in the presence of 100 nM BafA1 for 3 h and subjected to the HT-LC3 assay. Scale bars: 10 μm. MIL, Alexa Fluor 488-conjugated membrane-impermeable HT ligand; MPL, tetramethylrhodamine (TMR)-conjugated membrane-permeable HT ligand. (F) Dot plots of the cytoplasmic fluorescence intensities of MPL and MIL relative to the mean of GFP-WT-expressing cells in (E) ( n = 45). (G) Immunoblot analysis of lysates (input) and immunoprecipitates (IP) from the indicated U-2 OS cells. (H) Immunoblot analysis of U-2 OS cells that were starved in the presence or absence of 100 nM BafA1 for 3 h. (I) Bar plots of LC3-II and p62 degradation ratios in (H). (J) Confocal images of U-2 OS cells that were starved in the presence of 100 nM BafA1 for 3 h and subjected to the HT-LC3 assay. Scale bars: 10 μm (K) Dot plots of the cytoplasmic fluorescence intensities of MPL and MIL relative to the mean of GFP-WT-expressing cells in (J) ( n = 40). In (D), (F), (I), and (K), statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test. All values in the graphs are mean ± SD. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns, not significant.

Article Snippet: Rabbit polyclonal -p62 Ser349 , Cell Signaling Technology , Cat# 95697; RRID:AB_2800251.

Techniques: Ubiquitin Proteomics, Variant Assay, Western Blot, Stable Transfection, Transduction, Construct, Expressing, Membrane, Fluorescence

Journal: Cell reports

Article Title: Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

doi: 10.1016/j.celrep.2024.115016

Figure Lengend Snippet: (A) Schematic of the domain organization of the mouse VPS37A protein, gene locus, and targeting strategy. (B) Immunoblot analysis of primary mouse embryonic fibroblasts (MEFs) isolated from E13.5 embryos. (C) Bar plots of LC3-II and p62 levels relative to the mean of WT/WT MEFs in (B) ( n = 3). (D) Immunoblot of lysates (input) and IPs from VPS37A KO U-2 OS cells expressing the indicated plasmids. (E) Confocal images of HT-LC3-expressing immortalized MEFs that were starved in the presence of 100 nM BafA1 for 3 h and subjected to the HT-LC3 assay. Scale bars: 10 μm and 1 μm (magnified images). (F) Dot plots of the cytoplasmic fluorescence intensities of MIL and MPL relative to the mean of WT/WT MEFs starved in the presence of BafA1 for 3 h in (E) ( n = 50). (G) Electron micrographs of MEFs. Asterisks and a double asterisk indicate immature autophagic structures including phagophores and an autolysosome-like structure, respectively. Scale bars: 1 μm. (H) Bar plot of the number of autophagic structures per cytoplasmic area in (G) ( n = 21). (I) Immunoblot analysis of immortalized MEFs that were stably transduced with the HT-mGFP bulk autophagic flux reporter, pulse-labeled with MPL for 20 min, and starved for 6 h in the presence or absence of 100 nM BafA1. (J) Bar plot of HT/(HT-mGFP + HT) ratio relative to WT/WT MEFs in (I) ( n = 3). (K) Immunoblot analysis of MEFs that were serum starved for 4 h, pretreated with 10 μg/mL cycloheximide (CHX) and treated with 100 ng/mL EGF in the presence of CHX for the indicated durations. (L) Bar plot of the EGFR levels relative to respective α-tubulin in MEFs in (K) ( n = 3). Statistical significance was determined by Student’s t test (C and H), two-way ANOVA followed by Tukey’s multiple-comparisons test (F and L), and one-way ANOVA followed by Tukey’s multiple-comparisons test (J). All values in the graphs are mean ± SD. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: Rabbit polyclonal -p62 Ser349 , Cell Signaling Technology , Cat# 95697; RRID:AB_2800251.

Techniques: Western Blot, Isolation, Expressing, Fluorescence, Stable Transfection, Transduction, Labeling

Journal: Cell reports

Article Title: Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

doi: 10.1016/j.celrep.2024.115016

Figure Lengend Snippet: (A) Confocal images of MEFs that were starved for 3 h and stained for p62 and LC3. Scale bars: 10 μm. (B and C) Dot plots of the total p62 area (μm 2 ) (B) and global Pearson’s correlation coefficient of LC3 with p62 (C) in (A) ( n = 3). (D) Immunoblot analysis of 1% Triton X-100 (TX-100)-soluble and -insoluble fractions prepared from MEFs. (E) Bar plots of total p62, phosphorylated p62 (S405 and S351), and KEAP1 in (D) ( n = 3). In (B), (C), and (E), statistical significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple-comparisons test. All values in the bar graphs are mean ± SD. In (B) and (C), each dot represents the mean of each experiment ( n = 20 cells per experiment). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Rabbit polyclonal -p62 Ser349 , Cell Signaling Technology , Cat# 95697; RRID:AB_2800251.

Techniques: Staining, Western Blot

Journal: Cell reports

Article Title: Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

doi: 10.1016/j.celrep.2024.115016

Figure Lengend Snippet: (A) Confocal images of the VPS37A KO U-2 OS cells transduced with FLAG-APEX2 control or FLAG-APEX2-LC3B, preincubated with biotin phenol and H 2 O 2 , followed by DyLight 650-conjugated NeutrAvidin incubation, and stained for FLAG and p62. Scale bars: 10 μm and 1 μm (magnified images). (B) Volcano plot of proteins enriched by FLAG-APEX2-LC3B WT vs. FLAG-APEX2 control. Significantly enriched proteins are colored ( p < 0.05). (C) Immunoblot analysis of MEFs treated with 10 μM GSK8612 for the indicated periods of time. (D) Immunoblot analysis of MEFs that were transiently transfected with TBK1 siRNA (siTBK1) or control non-targeting siRNA (siNT) for 72 h. (E) Bar plots of the indicated protein levels relative to the siTBK1-transfected mut/mut MEFs in (D) ( n = 3). (F) Immunoblot analysis of MEFs that were stably transduced in combination with Cas9 and either non-targeting or TBK1 single guide RNAs. (G) Confocal images of HT-LC3-expressing MEFs treated with 10 μM GSK8612 for 48 h, pre-stained with MPL-TMR for 15 min, and stained for p62 and p-TBK1. Scale bars: 10 μm and 1 μm (magnified images). (H) Bar plot of total p62 area (μm 2 ) in (G) ( n = 3). (I) Immunoblot analysis of 1% TX-100-soluble and -insoluble fractions prepared from mut/mut MEFs incubated in complete medium in the presence or absence of 10 μM GSK8612 for 48 h. p62 levels in TX-100-soluble and TX-100-insoluble fractions relative to untreated MEFs are shown. (J) Confocal images of 8-week-old mouse liver sections stained for p62 and p -TBK1. Scale bars: 10 μm and 1 μm (magnified images). Statistical significance was determined by one-way ANOVA followed by Holm-Sidak’s multiple-comparisons test (E) and Mann-Whitney nonparametric t test (H). All values in the bar graphs are mean ± SD. In (H), each dot represents the mean of each experiment ( n = 20 cells per experiment). *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Rabbit polyclonal -p62 Ser349 , Cell Signaling Technology , Cat# 95697; RRID:AB_2800251.

Techniques: Transduction, Control, Incubation, Staining, Western Blot, Transfection, Stable Transfection, Expressing, MANN-WHITNEY

Journal: Cell reports

Article Title: Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain

doi: 10.1016/j.celrep.2024.115016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal -p62 Ser349 , Cell Signaling Technology , Cat# 95697; RRID:AB_2800251.

Techniques: Ubiquitin Proteomics, Recombinant, Modification, Membrane, Electron Microscopy, Protease Inhibitor, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Plasmid Preparation, Software, Imaging