Journal: Cell Death & Disease

Article Title: Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells

doi: 10.1038/s41419-022-05183-z

Figure Lengend Snippet: A Representative TEM images of TTM-treated HUVECs. Cells were treated with 100 μM TTM for 12 h. N, nucleus; C, cytoplasm; Mt, mitochondria; Yellow arrow, autophagosomes. B Representative immunofluorescence images of LC3B puncta in HUVECs treated with 30, 100, or 300 μM TTM for 12 h. Scale bar, 20 μm; Zoom-in scale bar, 10 μm. C Western blot analysis and quantification of LC3B and SQSTM1 in HUVECs treated with 100 μM TTM for 0, 3, 6, 9, and 12 h, respectively. β-Actin was used as loading control. D Western blot analysis and quantification of LC3B and SQSTM1 in TTM-treated HUVECs with or without lysosome inhibitor CQ (5 μM) and BafA1 (100 nM), respectively. GAPDH was used as loading control. E Autophagic flux assay using tandem fluorescent-tagged LC3B (RFP-GFP-LC3B) construct. Stable cell line expressing RFP-GFP-LC3B was pretreated with or without Baf A1 for 1 h and then co-incubated with TTM for 12 h. Scale bar, 20 μm; Zoom-in scale bar, 10 μm.

Article Snippet: The following primary antibodies were used: NRF2 (#16396-1-AP, 1:1,000, Proteintech), KEAP1 (#8047S, 1:3,000, Cell Signaling Technology), HMOX1 (#66743-1-Ig, 1:3,000, Proteintech), GCLM (#A5939, 1:3,000, Bimake), SLC7A11 (#ab175186, 1:1,000, Abcam) LC3B (#L7543, 1:3,000, Sigma), SQSTM1 (#18420-1-AP, 1:3,000, Proteintech), p-SQSTM1 (Ser349) (#16177, 1:1000, Cell Signaling Technology), ATG5 (#9980S, 1:3,000, Cell Signaling Technology), p-mTOR (Ser2448) (#5536S, 1:1000, Cell Signaling Technology), p-AMPKα (Thr172) (#2535T, 1:1,000, Cell Signaling Technology), p-ULK1 (Ser757) (#14202T, 1:1,000, Cell Signaling Technology), Cleaved PARP (#5625, 1:1,000, Cell Signaling Technology), γH2AX (#9718, 1:3,000, Cell Signaling Technology), GAPDH (#60004-1-Ig, 1:6,000, Proteintech) and β-Actin (#HC201-01, 1:10,000, TransGen).

Techniques: Immunofluorescence, Western Blot, Control, Flux Assay, Construct, Stable Transfection, Expressing, Incubation

Journal: Cell Death & Disease

Article Title: Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells

doi: 10.1038/s41419-022-05183-z

Figure Lengend Snippet: A Western blot analysis and quantification of NRF2, HMOX1, GCLM, ATG5, and LC3B in WT or ATG5 -KO cells treated with or without 100 μM TTM for 12 h. GAPDH was used as loading control. B qPCR analysis for HMOX1 , GCLM , and SLC7A11 in WT and ATG5 -KO cells after treatment with or without TTM. One-way ANOVA followed by a Tukey multiple comparison test was used for statistical analysis. * p < 0.05. C Western blot analysis and quantification of p-SQSTM1 (S349) and SQSTM1 in HUVECs treated with 100 μM TTM for indicated time. β-Actin was used as loading control. D Western blot analysis and quantification of KEAP1, NRF2, HMOX1, GCLM, and SQSTM1 in WT and SQSTM1 -KO cells treated with TTM for indicated time. β-Actin was used as loading control. E Western blot analysis and quantification of p-AMPKα (T172), p-MTOR (S2488), and p-ULK1 (S757) in TTM-treated HUVECs. β-Actin was used as loading control.

Article Snippet: The following primary antibodies were used: NRF2 (#16396-1-AP, 1:1,000, Proteintech), KEAP1 (#8047S, 1:3,000, Cell Signaling Technology), HMOX1 (#66743-1-Ig, 1:3,000, Proteintech), GCLM (#A5939, 1:3,000, Bimake), SLC7A11 (#ab175186, 1:1,000, Abcam) LC3B (#L7543, 1:3,000, Sigma), SQSTM1 (#18420-1-AP, 1:3,000, Proteintech), p-SQSTM1 (Ser349) (#16177, 1:1000, Cell Signaling Technology), ATG5 (#9980S, 1:3,000, Cell Signaling Technology), p-mTOR (Ser2448) (#5536S, 1:1000, Cell Signaling Technology), p-AMPKα (Thr172) (#2535T, 1:1,000, Cell Signaling Technology), p-ULK1 (Ser757) (#14202T, 1:1,000, Cell Signaling Technology), Cleaved PARP (#5625, 1:1,000, Cell Signaling Technology), γH2AX (#9718, 1:3,000, Cell Signaling Technology), GAPDH (#60004-1-Ig, 1:6,000, Proteintech) and β-Actin (#HC201-01, 1:10,000, TransGen).

Techniques: Western Blot, Control, Comparison

Journal: Cell Death & Disease

Article Title: Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells

doi: 10.1038/s41419-022-05183-z

Figure Lengend Snippet: A Representative morphological changes of WT control, ATG5 -KO or SQSTM1 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 24 h, respectively. Scale bar, 100 μm. B , C FACS analysis and quantification of DHE intensity in WT control or ATG5 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 12 h. D , E FACS analysis and quantification of DHE intensity in WT control or SQSTM1 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 12 h. In C and E , MFI mean fluorescence intensity. One-way ANOVA followed by a Tukey multiple comparison test was used for statistical analysis. * p < 0.05. F FACS analysis of apoptotic cell death in WT control, ATG5 -KO and SQSTM1 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 24 h. G Western blot analysis and quantification of HMOX1, cleaved PARP, γH2AX, and β-Actin in WT and ATG5 -KO cells treated with NaAsO 2 with or without TTM. H Western blot analysis and quantification of HMOX1, cleaved PARP, γH2AX and β-Actin in WT control or SQSTM1 -KO cells treated with NaAsO 2 with or without TTM.

Article Snippet: The following primary antibodies were used: NRF2 (#16396-1-AP, 1:1,000, Proteintech), KEAP1 (#8047S, 1:3,000, Cell Signaling Technology), HMOX1 (#66743-1-Ig, 1:3,000, Proteintech), GCLM (#A5939, 1:3,000, Bimake), SLC7A11 (#ab175186, 1:1,000, Abcam) LC3B (#L7543, 1:3,000, Sigma), SQSTM1 (#18420-1-AP, 1:3,000, Proteintech), p-SQSTM1 (Ser349) (#16177, 1:1000, Cell Signaling Technology), ATG5 (#9980S, 1:3,000, Cell Signaling Technology), p-mTOR (Ser2448) (#5536S, 1:1000, Cell Signaling Technology), p-AMPKα (Thr172) (#2535T, 1:1,000, Cell Signaling Technology), p-ULK1 (Ser757) (#14202T, 1:1,000, Cell Signaling Technology), Cleaved PARP (#5625, 1:1,000, Cell Signaling Technology), γH2AX (#9718, 1:3,000, Cell Signaling Technology), GAPDH (#60004-1-Ig, 1:6,000, Proteintech) and β-Actin (#HC201-01, 1:10,000, TransGen).

Techniques: Control, Fluorescence, Comparison, Western Blot

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: p62 is crucial for cell proliferation and promotes glucose metabolism. (A) Generation of p62-knockdown ACHN cells. ACHN cells were infected with lentiviruses expressing shRNA against p62 (sh-p62; two different oligonucleotides) or LacZ (sh-LacZ). Immunoblotting was performed to evaluate the expression of p62. GAPDH is an internal control. (B) A CCK-8 kit was utilized to quantify cell viability at indicated time points. Data presented are means±s.d. from three independent experiments. (C,D) Knockdown of p62 reduces glucose uptake under both normoxia and hypoxia in ACHN and Caki-1 cells. ACHN (C) or Caki-1 (D) cells stably expressing sh-p62-1#, sh-p62-2# or sh-LacZ were grown for 48 h. The cells were then subjected to normoxic or hypoxic conditions for 12 h, and glucose uptake was measured and expressed as a ratio of the levels in sh-LacZ cells. **Significant differences, P <0.01 (Student's t -test). (E,F) Cells were treated as in C, and extracellular lactate in the culture medium was measured and expressed as a ratio of the levels in sh-LacZ cells. ** P <0.01. (G) Knockdown of p62 decreases the expression of genes involved in glucose metabolism. The relative expression of genes (HIF1α, ENO1, PKM2, GLUT1, PDK1, LDHA and NDUFA4L2) involved in glucose metabolism in ACHN cells expressing sh-p62-1#, sh-p62-2# or sh-LacZ was measured using qRT-PCR analysis. Expression was normalized to that in cells expressing sh-LacZ (control); data are plotted as the mean±s.d. of three independent experiments. ** P <0.01 versus control (Student's t -test).

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Knockdown, Infection, Expressing, shRNA, Western Blot, Control, CCK-8 Assay, Stable Transfection, Quantitative RT-PCR

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: p62 is a positive regulator of HIF1α. (A,B) p62 knockdown reduces HIF1α mRNA without affecting HIF2α mRNA in cells both under normoxia and hypoxia. The relative expression of HIF1α and HIF2α in ACHN cells expressing sh-p62-1#, sh-p62-2# or sh-LacZ under normoxia and hypoxia was measured by using qRT-PCR analysis. Expression was normalized to that in cells expressing sh-LacZ (control); data are plotted as the mean±s.d. of three independent experiments. n.s., not significant ( P >0.05); * P <0.05 versus control; ** P <0.01 versus control (Student's t -test). (C) Relative expression of HIF-1α, GLUT1, PDK1, LDHA, VEGFα and NDUFA4L2 mRNA levels in control (LacZ), p62-knockdown or p62-rescued (sh-p62-1#+Flag-p62) ACHN cells, as determined by using RT-qPCR analysis. ** P <0.01 versus control; ## P <0.01 versus sh-p62-1#. (D) HRE–Fluc construct and luciferase activity measurement. ACHN cells were infected with lentivirus expressing 6×HRE-Fluc and SV40-Rluc (internal control, for normalization) luciferase reporter genes to generate HRE-reporter cells stably expressing firefly luciferase, controlled by HIF1. HRE-reporter cells were transiently transfected with Flag–HIF1α (0, 1 or 2 μg). After transfection for 36 h, luciferase fluorescence was measured and normalized to that of the internal control. Western blotting analysis of the whole-cell lysates from ACHN cells was performed to evaluate the expression of Flag-HIF1a. GAPDH is an internal control. ** P <0.01 versus control (Student's t -test); n =3. (E) p62 promotes 6×HRE-driven luciferase activity that is dependent on HIF1α in ACHN cells. HRE-reporter ACHN cells stably expressing sh-p62 were infected with different combinations of lentivirus encoding psi-Flag, Flag–p62, sh-LacZ and sh-HIF1α. After 72 h of culture, luciferase fluorescence was measured, and the ratio of Fluc to Rluc activity was normalized to that of cells expressing psi-Flag control plasmids (mean±s.d.; n =3). Western blotting analysis was performed to evaluate the expression of Flag-p62. GAPDH is an internal control. ** P <0.01 versus control (Student's t -test).

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Control, Construct, Luciferase, Activity Assay, Infection, Stable Transfection, Transfection, Fluorescence, Western Blot

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: p62-dependent HIF1α levels are regulated by mTORC1 and NF-κB. (A) Stable p62 knockdown in ACHN cells downregulates the phosphorylation of P70S6K. Immunoblot for p62, p-P70S6K, p70S6K and GAPDH protein levels in whole-cell lysates from control (sh-LacZ) or p62-knockdown (sh-p62-1# or 2#) ACHN cells. (B) Rapamycin treatment reduces HIF1α protein levels in p62-rescued (re-expressed) ACHN cells. ACHN cells stably expressing sh-p62-1# were infected with lentivirus encoding psi-Flag or Flag–p62. After 72 h of culture, cells were treated with (+) or without (−) 20 nM of rapamycin for 24 h before cell lysis. Cell lysates were subjected to SDS­PAGE followed by immunoblotting with antibodies against p62, p-P70S6K, p70S6K and GAPDH. Because the HIF1α protein is unstable under normoxia, HIF1α was enriched through immunoprecipitation with an anti-HIF1α (rabbit polyclonal) antibody, and then the protein levels of HIF1α were determined by western blotting. (C) Rapamycin treatment downregulates p62-mediated enhancement of 6×HRE-driven luciferase activity. HRE-reporter ACHN cells were infected with lentivirus encoding sh-p62-1#. After 72 h of culture, the cells were transiently transfected with Flag–p62 and treated with or without rapamycin (20 nM) for 24 h, followed by measurement of the luciferase activity. ** P <0.01 versus control (Student's t -test); mean±s.d.; n =3. (D) Knockdown of p65 reduces HIF1α protein levels in p62-rescued (re-expressed) ACHN cells. p62-knockdown cells were infected with different combinations of lentivirus that encoded Flag–p62 or/and sh-p65. After 72 h of culture, cell lysates were subjected to SDS­PAGE followed by immunoblotting with anti-­p62 and anti-­GAPDH antibodies. HIF1α was enriched through immunoprecipitation with anti-HIF1α (rabbit polyclonal) antibodies, and then the protein levels of HIF1α were determined by western blotting. (E) Knockdown of p65 downregulates p62-mediated enhancement of 6×HRE-driven luciferase activity. HRE-reporter ACHN cells were infected with lentivirus encoding sh-p62-1#. Cells were infected with different lentiviruses as in D. After 72 h of culture, the HRE luciferase activity was measured. ** P <0.01 versus control (Student's t -test); mean±s.d.; n =3. (F) Inhibition of NF-κB signaling reduces p62-mediated upregulation of HIF1α and the mRNA levels of its target genes. The relative expression of HIF1α and GLUT1 in p62-knockdown stable ACHN cells that had been transfected with different combinations of Flag–p62, psi-Flag and/or sh-p65. Expression was normalized to that in cells expressing psi-Flag (control). ** P <0.01 versus control Student's t -test); mean±s.d.; n =3.

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Stable Transfection, Expressing, Infection, Lysis, Immunoprecipitation, Luciferase, Activity Assay, Transfection, Inhibition

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: p62 interacts with the VHL E3 ubiquitin ligase complex. (A) p62 interacts with CUL2. Co-transfection of Myc–CUL2 into HEK293T cells was performed, together with Flag–VHL or Flag–p62. At 48 h after transfection, the whole-cell lysates were extracted for co-immunoprecipitation with an anti-Flag antibody, followed by probing for Myc. IB, immunoblotting; IP, immunoprecipitation. (B) HEK293T cells were co-transfected with HA–p62 together with Flag–VHL, Flag–CUL2, Flag–elonginB, Flag–elonginC, Flag–HIF1α or psi-Flag. At 48 h after transfection, the whole-cell lysates were extracted for co-immunoprecipitation with an antibody against Flag, followed by probing with an anti-HA antibody. (C) p62 interacts with VHL and CUL2 in vivo . The HEK293T cell lysates were immunoprecipitated (IP) with a control antibody (rabbit IgG) or an anti-­p62 antibody, and analyzed by immunoblotting (IB) with antibodies against VHL, CUL2 or HIF1α. (D) Domain structure and deletion constructs of VHL (left) and p62 (right). Numbers refer to amino acid residues. (E) Mapping of the p62 ­binding region of VHL. HEK293T cells were transiently transfected with HA–p62 along with various Flag-­tagged VHL deletion mutants, as indicated. The cell lysates were immunoprecipitated with an anti-­Flag antibody and immunoblotted with an anti-­HA antibody. (F) Mapping of the VHL- and CUL2-binding region of p62. HEK293T cells were transiently transfected with HA–VHL and Myc–CUL2 along with various Flag-tagged p62 deletion mutants, as indicated. The cell lysates were immunoprecipitated with anti-­Flag antibody and immunoblotted with anti-­HA and anti-Myc antibodies.

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Ubiquitin Proteomics, Cotransfection, Transfection, Immunoprecipitation, Western Blot, In Vivo, Control, Construct, Binding Assay

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: Colocalization of p62 with CUL2 and VHL. (A–E) Caki-1 cells were transfected with different combinations of expression vectors with pmCherry-C1 (control), mCherry–VHL, mCherry–CUL2, pEGFP-C1 and EGFP–p62. After transfection for 48 h, the cells were treated with Hoechst 33258 to stain the nucleus and observed by using confocal microscopy. Arrows indicate colocalization in aggregates. Scale bars: 10 µm.

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Transfection, Expressing, Control, Staining, Confocal Microscopy

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: Interaction with p62 is required for VHL and p62 aggregation. (A,B) Generation of p62-knockdown cells that were engineered to re-express p62–EGFP, p62Δ30-102–EGFP or p62Δ170-260–EGFP. HEK293T cells were infected with lentivirus expressing shRNA against p62 (sh-p62-1#), and then p62–EGFP, p62Δ30-102–EGFP or p62Δ170-260–EGFP was expressed in the knockdown cells to establish stably expressing cell lines. (A) Images were taken with a fluorescence microscope. Scale bars: 10 µm. (B) Immunoblotting of lysates derived from these engineered cells, using antibodies against p62 and GAPDH. (C–H) The engineered cell lines were infected with lentivirus encoding mCherry–VHL or (F–H) mCherry–CUL2. After transfection for 48 h, the cells were stained with Hoechst 33258 and observed by using confocal microscopy. Arrows indicate colocalization in aggregates. Scale bars: 5 µm.

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Knockdown, Infection, Expressing, shRNA, Stable Transfection, Fluorescence, Microscopy, Western Blot, Derivative Assay, Transfection, Staining, Confocal Microscopy

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: p62 stabilizes HIF1α by negatively regulating VHL ubiquitin ligase activity. (A) p62 increases the HIF1α protein levels. HEK293T cells were co-transfected with Flag–mCherry (0.2 μg), Flag–HIF1α (0.2 μg) and p62–EGFP–Myc (0, 0.2 or 0.6 μg) plasmids. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-Flag and anti-Myc antibodies. (B) The VHL- and CUL2-binding region of p62 is necessary for p62-mediated increases of HIF1α protein levels. HEK293T cells were co-transfected with Flag–mCherry (0.2 μg), Flag–HIF1α (0.2 μg) together with Myc–VHL (0, 0.2 or 0.6 μg), p62Δ170-260–EGFP–Myc (0.6 μg) or p62–EGFP–Myc (0.6 μg). Cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-Flag and anti-Myc antibodies. (C) p62 increases the stability of the endogenous HIF1α protein. ACHN cells expressing p62–EGFP–Myc or parental ACHN cells were incubated under hypoxia (12 h) and then moved to normoxia (time 0) with the addition of cycloheximide (CHX, 50 μg/ml). Cell lysates were subsequently harvested at sequential time points (0, 2, 5 or 10 min) after treatment, and then the cell lysates were immunoblotted for HIF1α, Myc or GAPDH. (D) p62 binds to the VHL complex and competes with HIF1α. HEK293T cells were co-transfected with Flag–p62, Myc–CUL2, HA–VHL and EGFP–HIF1α (0, 0.4 or 1 μg) plasmids. Cell lysates were precipitated (IP) with anti-Flag antibody and immunoblotted (IB) for Myc and HA. (E) p62 attenuates the interaction between VHL and HIF1α. HEK293T cells were co-transfected with HA–VHL, Flag–HIF1α and/or p62–EGFP–Myc plasmids. At 24 h post transfection, cells were maintained under normoxia or exposed to hypoxia for 12 h. Cell lysates were precipitated with an anti-HA antibody and immunoblotted for Flag. (F) p62 competes with DCNL1 for binding to CUL2. HEK293T cells were transfected with Myc–CUL2 and Flag–DCNL1 together with an increasing amount of HA–p62 (0, 0.4 or 1 μg). Cell lysates were precipitated with anti-Flag antibody and immunoblotted for Myc. (G) p62 attenuates NEDD8-mediated modification of CUL2. HEK293T cells were co-transfected with Myc–CUL2 and HA–NEDD8, and an increasing amount of Flag–p62. Cell lysates were immunoprecipitated with an anti-Myc antibody and immunoblotted with the indicated antibodies. (H) p62 inhibits endogenous ubiquitylation of both HIF1α and HIF2α. HEK293T cells were infected with lentivirus expressing His–HA–ubiquitin (6×His-HA-UB). After 72 h in culture, cells were treated with 10 µM MG-132 for 12 h, and subsequently protein lysates were prepared in urea buffer. Ni-NTA affinity purifications were used to pull down only proteins covalently modified with 6×His-HA-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were immunoblotted for HA (ubiquitin), HIF1α, HIF2α and p62.

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Ubiquitin Proteomics, Activity Assay, Transfection, SDS Page, Western Blot, Binding Assay, Expressing, Incubation, Modification, Immunoprecipitation, Infection, Purification

Journal: Journal of Cell Science

Article Title: Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

doi: 10.1242/jcs.178756

Figure Lengend Snippet: HIF1α contributes to the function of p62 in regulating the Warburg effect and growth in soft agar. (A) Total protein was extracted from ACHN cells that stably expressed sh-p62 and/or sh-HIF1α, followed by western blotting to evaluate the expression of HIF1α. GAPDH is an internal control. (B,C) Knockdown of HIF1α severely impairs p62-mediated increased glucose uptake and lactate production. ACHN cells that stably expressed sh-p62-1# and/or sh-HIF1α were infected with lentivirus expressing Flag–p62. After transfection for 72 h, glucose uptake (B) and extracellular lactate in the culture medium (C) were measured and expressed as a ratio of those in sh-LacZ cells. Significant differences, ** P <0.01 (Student's t -test); mean±s.d. n =3. (D) Immunoblotting for HIF1α, Flag and GAPDH (loading control) of lysates derived from ACHN cells that stably expressed sh-p62 and/or sh-HIF1α and that had then been infected with lentivirus expressing Flag–HIF1α. (E,F) The effect of silencing p62 on glucose uptake and lactate production was reversed by HIF1α overexpression. Cells were treated as described in D. (E) Glucose uptake and (F) extracellular lactate in the culture medium were measured. ** P <0.01 Student's t -test); mean±s.d. n =3. (G,H) Generation of ACHN cells that stably expressed shRNA against p62 (sh-p62-1#) or against HIF1α (sh-HIF1α) (G), which were then infected with lentivirus encoding psi-Flag (Control), Flag–p62 or Flag–HIF1α (H). Western blotting analysis of the lysates from these engineered cells was performed to evaluate the expression of HIF1α, p62, Flag–p62 and Flag–HIF1α. GAPDH is an internal control. (I) p62 regulates colony growth in soft agar through HIF1α. ACHN cells in which p62 or p62 and HIF1α had been knocked down were infected with lentivirus expressing psi-Flag (control), Flag–p62 or Flag–HIF1α. After transfection for 48 h, the cells were plated in soft agar. The number of colonies was measured after 14 days. (Ia–Ih) Representative photographs were taken by using an inverted microscope. (J) The number of colonies was quantified in three random images from each treatment group. Results are the mean±s.d. from three independent experiments, plotted as the percentage of colonies formed by sh-LacZ-expressing cells. Significant differences, ** P <0.01 (Student's t -test).

Article Snippet: Anti-p62 (#8025, 1:1000), anti-HIF1α (#3716, 1:200), anti-p65 (#6956, 1:1000), anti-p70S6K (#9202, 1:1000), anti-phospho-p70S6K (Thr389) (#9205, 1:1000), anti-VHL (#2738, 1:500) antibodies were from Cell Signaling Technology; anti-HIF1α (NB100-105, 1:500) and anti-HIF2α (NB100-122, 1:500) antibodies were from Novus Biologicals (Littleton, CO); anti-VHL (clone D-7) antibody was from Santa Cruz Biotechnology; anti-GAPDH (CW0100, 1:2000) antibody was purchased from Beijing CWBio (CWBIO, Peking, China); goat anti- mouse IgG horseradish peroxidase (HRP)- conjugated whole antibody (31430, 1:50,000) and goat anti- rabbit IgG-HRP- conjugated whole antibody (31460, 1:50,000) was purchased from Thermo Scientific.

Techniques: Stable Transfection, Western Blot, Expressing, Control, Knockdown, Infection, Transfection, Derivative Assay, Over Expression, shRNA, Inverted Microscopy

Journal: Molecular cell

Article Title: Acetyl-CoA Derived from Hepatic Peroxisomal β-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation

doi: 10.1016/j.molcel.2020.05.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-p62 , Cell Signaling Technology , Cat# 5114; RRID:AB_10624872.

Techniques: Virus, Plasmid Preparation, Recombinant, Protease Inhibitor, Expressing, Construct, Generated, Software

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: Characterization of the effect of the ZIKV NS5 protein on microtubule stabilization and reorganization and on the accumulation of the autophagy marker p62. ( a ) Immunofluorescence microscopy analysis of the effect of NS5 on microtubules (MTs) in HEK-293T cells overexpressing NS5-myc (1.5 µg cDNA) compared to that in control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA). In this representative experiment, cells were processed for immunostaining with antibodies against the acetylated Lys 40 residue in α-tubulin (green) and/or NS5 (using an anti-myc mAb) (red). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 40× objective and analysed with the MetaMorph program. ( b ) Immunofluorescence microscopy images showing the exacerbated nested structure of acetylated MTs promoted in cells overexpressing NS5 (1.5 µg cDNA) (images captured using a 100× objective). As a control for the acetylation of MTs, cells were treated with tubacin (1 μM), a specific inhibitor of the deacetylase activity of HDAC6. This image (captured using a 40× objective) also serves as a control for the NS5-mediated acetylation of MTs images presented in panel ( a ). In these representative experiments, cells were processed for immunostaining with antibodies against the acetylated Lys 40 residue in α-tubulin (green) and/or NS5 (using an anti-myc mAb) (red). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100× or 40× objective, as indicated, and analysed with the MetaMorph program. ( c ) Quantitative Western blot analysis of the dose–response effect of NS5 overexpression on MT stabilization, as monitored by the acetylation of MTs at Lys 40 α-tubulin residues. The α-tubulin protein was used as the loading control protein. A representative of nine independent experiments is shown (see associated data in ). Histograms quantifying the amount of acetylated α-tubulin induced by NS5 in stabilized MTs from nine replicates of the experiment shown in panel ( c ). The data were normalized to the total α-tubulin protein. ( d ) Western blot analysis of the effect of the NS5 viral protein (1.5 µg cDNA) on the accumulation of the autophagic protein p62 in HEK-293T cells. NS5, p62 and total α-tubulin are shown. Histograms quantifying the amount of p62 stabilized by NS5 from the experiments are shown in panel ( d ). The data were normalized to the total α-tubulin protein. The data are presented as the means ± S.E.M. of three independent experiments (see associated data in ). ( e ) Quantitative Western blot analysis of the dose–response effect of NS5 overexpression on the accumulation of the autophagic protein p62 in HEK-293T cells overexpressing NS5. NS5, p62 and total α-tubulin are shown. The data are presented as the means ± S.E.M. of four independent experiments (see associated data in ). In panels ( c – e ), when indicated, the p values are **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.1; ns indicates not statistically significant ( p > 0.05). The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: Mouse anti-myc (9E10; sc-40), rabbit anti-HDAC6 (H-300; sc-11420), rabbit anti-HA (Y-11; sc-805), mouse anti-p62/SQSTM1 (D3; sc-28359) and mouse anti-Histone 3 (1G1; sc-517576) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Marker, Immunofluorescence, Microscopy, Control, Transfection, Plasmid Preparation, Immunostaining, Residue, Staining, Histone Deacetylase Assay, Activity Assay, Western Blot, Over Expression, Comparison

Journal: Cells

Article Title: The ZIKV NS5 Protein Aberrantly Alters the Tubulin Cytoskeleton, Induces the Accumulation of Autophagic p62 and Affects IFN Production: HDAC6 Has Emerged as an Anti-NS5/ZIKV Factor

doi: 10.3390/cells13070598

Figure Lengend Snippet: HDAC6 promotes the autophagic clearance of the ZIKV NS5 protein. ( a ) Quantitative Western blot analysis of HEK-293T cells overexpressing NS5-myc (lane 2) or the N-terminal HA-tagged HDAC6 mutant lacking the C-terminal BUZ domain (HA-HDAC6-DBUZ) (lane 3) or both constructs (lane 4) (1.5 µg cDNA) and comparison to control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) (lane 1). The absence of a degradative effect of HA-HDAC6-DBUZ on NS5-myc expression (lane 4) and the deacetylation of MTs by HA-HDAC6-DBUZ, which is deacetylase active (lanes 3 and 4) are shown. NS5 could not stabilize acetylated MTs under these experimental conditions (lane 4). The α-tubulin protein was used as the loading control protein. A representative experiment of three is shown (see associated data in ). Left histograms quantifying the amount of NS5-myc expressed in the absence (corresponding to lane 2 in the western blot panel) or presence of overexpressed HA-HDAC6-DBUZ (corresponding to lane 4 in the western blot panel), normalized to total α-tubulin. Right histograms quantifying the amount of p62 protein expressed in cells overexpressing NS5-myc, HA-HDAC6-DBUZ or both constructs (corresponding to lanes 2–4 in the western blot panel, respectively) and compared with that in control cells (corresponding to lane 1 in the western blot panel). These data are presented as the means ± S.E.M. of three independent experiments. ( b ) Immunofluorescence microscopy analysis of NS5 cell distribution in cells treated or not treated (control) with 3-MA (5 mM), an inhibitor of HDAC6-mediated aggresome formation and further autophagy clearance of targeted proteins, in cells overexpressing NS5-myc (1.5 µg cDNA). The cells were processed for immunostaining with an anti-myc mAb (green). DAPI is a blue, fluorescent probe for DNA staining. Images were captured using a 100x objective and analysed with the MetaMorph program. ( c ) Quantitative Western blot analysis of HEK-293T cells overexpressing NS5-myc (lane 2), the functional HA-HDAC6 construct (lane 3) or both constructs (lane 4) (1.5 µg cDNA), compared to control cells transfected with the pcDNA TM 3.1(+) plasmid (1.5 µg cDNA) (lane 1) under control conditions (PBS-treated cells, vehicle for 3-MA) or cells treated with 3-MA (5 mM). The absence of a degradative effect exerted by HA-HDAC6 on NS5 in the presence of 3-MA (lane 4 in 3-MA-treated cells compared to lane 4 in PBS-treated cells) and the deacetylation of MTs by HA-HDAC6 are shown. The α-tubulin protein was used as the loading control protein. A representative experiment of 2 is shown (see associated data in ). Histograms quantifying the amount of NS5-myc expressed in the absence (lane 2) or presence of overexpressed HA-HDAC6 (lane 4), normalized to total α-tubulin, in both PBS- and 3-MA-treated cells are shown. The data are presented as the means ± S.E.M. of 2 independent experiments. ( d ) Quantitative Western blot analysis of the effect of siRNA-mediated knockdown of endogenous HDAC6 (siRNA-HDAC6, 25 pmol/well) on the level of NS5 protein expression in HEK-293T cells overexpressing NS5-myc and compared to that in control and scrambled (1 µM)-treated cells. A representative experiment of four is shown (see associated data in ). Histograms quantifying the amount of endogenous HDAC6 and NS5 detected in cells treated with siRNA-HDAC6 or scrambled oligos, normalized to total α-tubulin. The data are presented as the means ± S.E.M. of four independent experiments. For quantitative analysis of Western blots, when indicated, the p values are **** p ≤ 0.0001 and * p ≤ 0.1; ns indicates not statistically significant ( p > 0.05). The p value is the comparison of the means between the two groups using the parametric Student’s t test.

Article Snippet: Mouse anti-myc (9E10; sc-40), rabbit anti-HDAC6 (H-300; sc-11420), rabbit anti-HA (Y-11; sc-805), mouse anti-p62/SQSTM1 (D3; sc-28359) and mouse anti-Histone 3 (1G1; sc-517576) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Mutagenesis, Construct, Comparison, Control, Transfection, Plasmid Preparation, Expressing, Histone Deacetylase Assay, Immunofluorescence, Microscopy, Immunostaining, Staining, Functional Assay, Knockdown

Journal: Cell Death & Disease

Article Title: Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells

doi: 10.1038/s41419-022-05183-z

Figure Lengend Snippet: A Representative TEM images of TTM-treated HUVECs. Cells were treated with 100 μM TTM for 12 h. N, nucleus; C, cytoplasm; Mt, mitochondria; Yellow arrow, autophagosomes. B Representative immunofluorescence images of LC3B puncta in HUVECs treated with 30, 100, or 300 μM TTM for 12 h. Scale bar, 20 μm; Zoom-in scale bar, 10 μm. C Western blot analysis and quantification of LC3B and SQSTM1 in HUVECs treated with 100 μM TTM for 0, 3, 6, 9, and 12 h, respectively. β-Actin was used as loading control. D Western blot analysis and quantification of LC3B and SQSTM1 in TTM-treated HUVECs with or without lysosome inhibitor CQ (5 μM) and BafA1 (100 nM), respectively. GAPDH was used as loading control. E Autophagic flux assay using tandem fluorescent-tagged LC3B (RFP-GFP-LC3B) construct. Stable cell line expressing RFP-GFP-LC3B was pretreated with or without Baf A1 for 1 h and then co-incubated with TTM for 12 h. Scale bar, 20 μm; Zoom-in scale bar, 10 μm.

Article Snippet: The following primary antibodies were used: NRF2 (#16396-1-AP, 1:1,000, Proteintech), KEAP1 (#8047S, 1:3,000, Cell Signaling Technology), HMOX1 (#66743-1-Ig, 1:3,000, Proteintech), GCLM (#A5939, 1:3,000, Bimake), SLC7A11 (#ab175186, 1:1,000, Abcam) LC3B (#L7543, 1:3,000, Sigma), SQSTM1 (#18420-1-AP, 1:3,000, Proteintech), p-SQSTM1 (Ser349) (#16177, 1:1000, Cell Signaling Technology), ATG5 (#9980S, 1:3,000, Cell Signaling Technology), p-mTOR (Ser2448) (#5536S, 1:1000, Cell Signaling Technology), p-AMPKα (Thr172) (#2535T, 1:1,000, Cell Signaling Technology), p-ULK1 (Ser757) (#14202T, 1:1,000, Cell Signaling Technology), Cleaved PARP (#5625, 1:1,000, Cell Signaling Technology), γH2AX (#9718, 1:3,000, Cell Signaling Technology), GAPDH (#60004-1-Ig, 1:6,000, Proteintech) and β-Actin (#HC201-01, 1:10,000, TransGen).

Techniques: Immunofluorescence, Western Blot, Control, Flux Assay, Construct, Stable Transfection, Expressing, Incubation

Journal: Cell Death & Disease

Article Title: Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells

doi: 10.1038/s41419-022-05183-z

Figure Lengend Snippet: A Western blot analysis and quantification of NRF2, HMOX1, GCLM, ATG5, and LC3B in WT or ATG5 -KO cells treated with or without 100 μM TTM for 12 h. GAPDH was used as loading control. B qPCR analysis for HMOX1 , GCLM , and SLC7A11 in WT and ATG5 -KO cells after treatment with or without TTM. One-way ANOVA followed by a Tukey multiple comparison test was used for statistical analysis. * p < 0.05. C Western blot analysis and quantification of p-SQSTM1 (S349) and SQSTM1 in HUVECs treated with 100 μM TTM for indicated time. β-Actin was used as loading control. D Western blot analysis and quantification of KEAP1, NRF2, HMOX1, GCLM, and SQSTM1 in WT and SQSTM1 -KO cells treated with TTM for indicated time. β-Actin was used as loading control. E Western blot analysis and quantification of p-AMPKα (T172), p-MTOR (S2488), and p-ULK1 (S757) in TTM-treated HUVECs. β-Actin was used as loading control.

Article Snippet: The following primary antibodies were used: NRF2 (#16396-1-AP, 1:1,000, Proteintech), KEAP1 (#8047S, 1:3,000, Cell Signaling Technology), HMOX1 (#66743-1-Ig, 1:3,000, Proteintech), GCLM (#A5939, 1:3,000, Bimake), SLC7A11 (#ab175186, 1:1,000, Abcam) LC3B (#L7543, 1:3,000, Sigma), SQSTM1 (#18420-1-AP, 1:3,000, Proteintech), p-SQSTM1 (Ser349) (#16177, 1:1000, Cell Signaling Technology), ATG5 (#9980S, 1:3,000, Cell Signaling Technology), p-mTOR (Ser2448) (#5536S, 1:1000, Cell Signaling Technology), p-AMPKα (Thr172) (#2535T, 1:1,000, Cell Signaling Technology), p-ULK1 (Ser757) (#14202T, 1:1,000, Cell Signaling Technology), Cleaved PARP (#5625, 1:1,000, Cell Signaling Technology), γH2AX (#9718, 1:3,000, Cell Signaling Technology), GAPDH (#60004-1-Ig, 1:6,000, Proteintech) and β-Actin (#HC201-01, 1:10,000, TransGen).

Techniques: Western Blot, Control, Comparison

Journal: Cell Death & Disease

Article Title: Ammonium tetrathiomolybdate triggers autophagy-dependent NRF2 activation in vascular endothelial cells

doi: 10.1038/s41419-022-05183-z

Figure Lengend Snippet: A Representative morphological changes of WT control, ATG5 -KO or SQSTM1 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 24 h, respectively. Scale bar, 100 μm. B , C FACS analysis and quantification of DHE intensity in WT control or ATG5 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 12 h. D , E FACS analysis and quantification of DHE intensity in WT control or SQSTM1 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 12 h. In C and E , MFI mean fluorescence intensity. One-way ANOVA followed by a Tukey multiple comparison test was used for statistical analysis. * p < 0.05. F FACS analysis of apoptotic cell death in WT control, ATG5 -KO and SQSTM1 -KO cells treated with 30 μM NaAsO 2 with or without 100 μM TTM for 24 h. G Western blot analysis and quantification of HMOX1, cleaved PARP, γH2AX, and β-Actin in WT and ATG5 -KO cells treated with NaAsO 2 with or without TTM. H Western blot analysis and quantification of HMOX1, cleaved PARP, γH2AX and β-Actin in WT control or SQSTM1 -KO cells treated with NaAsO 2 with or without TTM.

Article Snippet: The following primary antibodies were used: NRF2 (#16396-1-AP, 1:1,000, Proteintech), KEAP1 (#8047S, 1:3,000, Cell Signaling Technology), HMOX1 (#66743-1-Ig, 1:3,000, Proteintech), GCLM (#A5939, 1:3,000, Bimake), SLC7A11 (#ab175186, 1:1,000, Abcam) LC3B (#L7543, 1:3,000, Sigma), SQSTM1 (#18420-1-AP, 1:3,000, Proteintech), p-SQSTM1 (Ser349) (#16177, 1:1000, Cell Signaling Technology), ATG5 (#9980S, 1:3,000, Cell Signaling Technology), p-mTOR (Ser2448) (#5536S, 1:1000, Cell Signaling Technology), p-AMPKα (Thr172) (#2535T, 1:1,000, Cell Signaling Technology), p-ULK1 (Ser757) (#14202T, 1:1,000, Cell Signaling Technology), Cleaved PARP (#5625, 1:1,000, Cell Signaling Technology), γH2AX (#9718, 1:3,000, Cell Signaling Technology), GAPDH (#60004-1-Ig, 1:6,000, Proteintech) and β-Actin (#HC201-01, 1:10,000, TransGen).

Techniques: Control, Fluorescence, Comparison, Western Blot

Journal: The Journal of cell biology

Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

doi: 10.1083/jcb.202501207

Figure Lengend Snippet: (A) Workflow of first proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of second proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of third proteomics experiments using the SHKBP1 F44A CUL3-binding deficient mutant to reduce CRL complex components in IP compared to SHKBP1 WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log 2 (fold changes of protein abundance in heavy/light samples) vs. statistical significance (−log 10 (p-value)). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in grey. Those above the cutoff with p values above 0.05 are shown in green, and those with p values below 0.05 are shown in purple.

Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

Techniques: Stable Transfection, Expressing, Construct, Binding Assay, Mutagenesis, Multiplex sample analysis, Quantitative Proteomics

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy

doi: 10.1080/15548627.2021.1874660

Figure Lengend Snippet: SQSTM1/p62 is a bona fide BPLF1 substrate. (A) Interaction of BPLF1 with the autophagy receptor protein SQSTM1/p62 was validated by reciprocal co-immunoprecipitation (IP). Western blots (WB) from representative experiments are shown. (B) Isotype control of SQSTM1/p62 immunoprecipitation. Weak non-specific binding of BPLF1 to the sepharose beads was observed with isotype control precipitation. (C) In vitro affinity isolation experiment shows direct binding of BPLF1 to SQSTM1/p62. (D) To resolve noncovalent protein interactions, endogenous SQSTM1/p62 was immunoprecipitated in denaturing conditions. Precipitated SQSTM1/p62 was free of ubiquitin in the presence of catalytically active BPLF1 and heavily ubiquitinated in the presence of the catalytic mutant. A non-specific 75 kDa band detected in total cell lysates by the anti-HA antibody is indicated by an asterisk. The antibody heavy chain is detected as a 50 kDa band in the IP blot. (E) Densitometry quantification of the ubiquitin smear in two independent experiments

Article Snippet: Antibodies and their manufacturers were: rat anti-BPLF1 [ ] (WB 1:1600, 2E5 supernatant) was from Helmholtz Zentrum (2E5); mouse anti-FLAG clone M2 (WB 1:4000, IF 1:400; F1804) and rabbit anti-LC3B (WB 1:1000, IF 1:200; L7543) were from Sigma-Aldrich; goat anti-FLAG (1:400; ab1257) and rabbit anti-SQSTM1/p62 (IP 1–2 μg/mg of lysate; ab101266), rabbit anti-GFP (WB 1:2000, ab290) and rabbit IgG isotype control (ab172730) were from Abcam; mouse anti-SQSTM1/p62 (WB 1:1000 IF 1:200; 610,832) was from BD Biosciences; rabbit anti-SQSTM1/p62 (WB 1:1000, 8025S) was from Cell Signaling Technology; mouse anti-HA clone 12 CA5 (WB 1:2000; 11,583,816,001) from Roche; mouse IgG isotype control (14–4732-82) was from Invitrogen; Alexa Fluor 488-, 555-, 594- and 647-conjugated secondary antibodies were from Thermo Fisher (A21206, A31570, A11032 and A21447, respectively).

Techniques: Immunoprecipitation, Western Blot, Control, Binding Assay, In Vitro, Isolation, Ubiquitin Proteomics, Mutagenesis

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy

doi: 10.1080/15548627.2021.1874660

Figure Lengend Snippet: Catalytic mutant BPLF1 promotes SQSTM1/p62 hyper-ubiquitination. (A) SQSTM1/p62 was immunoprecipitated in denaturing conditions from cells expressing increasing amounts of BPLF1 C61A and HA-tagged ubiquitin and western blots were probed with an anti-HA antibody. The ubiquitination of SQSTM1/p62 was increased in the presence of BPLF C61A in a dose dependent manner and reversed by spiking the transfections with catalytically active BPLF1. A non-specific 75 kD band detected in total cell lysates by the anti-HA antibody is indicated by an asterisk. (B) Quantification of the data presented in A. Mean ± SD of two experiments. (C) The hyper-ubiquitination of SQSTM1/p62 is largely due to addition of K48-linked ubiquitin chains. (D) Quantification of data presented in C; mean ± SD of two independent experiments

Article Snippet: Antibodies and their manufacturers were: rat anti-BPLF1 [ ] (WB 1:1600, 2E5 supernatant) was from Helmholtz Zentrum (2E5); mouse anti-FLAG clone M2 (WB 1:4000, IF 1:400; F1804) and rabbit anti-LC3B (WB 1:1000, IF 1:200; L7543) were from Sigma-Aldrich; goat anti-FLAG (1:400; ab1257) and rabbit anti-SQSTM1/p62 (IP 1–2 μg/mg of lysate; ab101266), rabbit anti-GFP (WB 1:2000, ab290) and rabbit IgG isotype control (ab172730) were from Abcam; mouse anti-SQSTM1/p62 (WB 1:1000 IF 1:200; 610,832) was from BD Biosciences; rabbit anti-SQSTM1/p62 (WB 1:1000, 8025S) was from Cell Signaling Technology; mouse anti-HA clone 12 CA5 (WB 1:2000; 11,583,816,001) from Roche; mouse IgG isotype control (14–4732-82) was from Invitrogen; Alexa Fluor 488-, 555-, 594- and 647-conjugated secondary antibodies were from Thermo Fisher (A21206, A31570, A11032 and A21447, respectively).

Techniques: Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Expressing, Western Blot, Transfection

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy

doi: 10.1080/15548627.2021.1874660

Figure Lengend Snippet: BPLF1 expression is associated with the formation of smaller SQSTM1/p62 aggregates. (A) Endogenous SQSTM1/p62 was detected by immunofluorescence in HeLa cells transfected with either empty vector, BPLF1 or BPLF1[C61A]. Scale bar: 10 μm. (B) Box and whiskers plot of the size of SQSTM1/p62 dots. A decrease in the median size of the SQSTM1/p62 dots was observed in the presence of catalytically active BPLF1. Median size: vector and BPLF1[C61A] = 0.140 μm; BPLF1 = 0.1 μm. (C) Small SQSTM1/p62 dots were overrepresented and large dots underrepresented in cells expressing the active viral enzyme. Figures D and E were constructed by pooling data from nine independent experiments

Article Snippet: Antibodies and their manufacturers were: rat anti-BPLF1 [ ] (WB 1:1600, 2E5 supernatant) was from Helmholtz Zentrum (2E5); mouse anti-FLAG clone M2 (WB 1:4000, IF 1:400; F1804) and rabbit anti-LC3B (WB 1:1000, IF 1:200; L7543) were from Sigma-Aldrich; goat anti-FLAG (1:400; ab1257) and rabbit anti-SQSTM1/p62 (IP 1–2 μg/mg of lysate; ab101266), rabbit anti-GFP (WB 1:2000, ab290) and rabbit IgG isotype control (ab172730) were from Abcam; mouse anti-SQSTM1/p62 (WB 1:1000 IF 1:200; 610,832) was from BD Biosciences; rabbit anti-SQSTM1/p62 (WB 1:1000, 8025S) was from Cell Signaling Technology; mouse anti-HA clone 12 CA5 (WB 1:2000; 11,583,816,001) from Roche; mouse IgG isotype control (14–4732-82) was from Invitrogen; Alexa Fluor 488-, 555-, 594- and 647-conjugated secondary antibodies were from Thermo Fisher (A21206, A31570, A11032 and A21447, respectively).

Techniques: Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Construct

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy

doi: 10.1080/15548627.2021.1874660

Figure Lengend Snippet: BPLF1 inhibits the recruitment of LC3 to SQSTM1/p62-positive structures. (A) HeLa cells expressing catalytically active or inactive BPLF1 or vector control were stained for endogenous SQSTM1/p62 and LC3. In cells expressing catalytically active BPLF1 a significant decrease of LC3 recruitment to SQSTM1/p62-positive structures was observed. (B) Quantification of the data presented in A. Mean ± SEM of the % LC3-positive SQSTM1/p62 dots detected in 14, 22 and 24 images pooled from two independent experiments. Statistical analysis was performed using Student t-test. ***P ≤ 0.001

Article Snippet: Antibodies and their manufacturers were: rat anti-BPLF1 [ ] (WB 1:1600, 2E5 supernatant) was from Helmholtz Zentrum (2E5); mouse anti-FLAG clone M2 (WB 1:4000, IF 1:400; F1804) and rabbit anti-LC3B (WB 1:1000, IF 1:200; L7543) were from Sigma-Aldrich; goat anti-FLAG (1:400; ab1257) and rabbit anti-SQSTM1/p62 (IP 1–2 μg/mg of lysate; ab101266), rabbit anti-GFP (WB 1:2000, ab290) and rabbit IgG isotype control (ab172730) were from Abcam; mouse anti-SQSTM1/p62 (WB 1:1000 IF 1:200; 610,832) was from BD Biosciences; rabbit anti-SQSTM1/p62 (WB 1:1000, 8025S) was from Cell Signaling Technology; mouse anti-HA clone 12 CA5 (WB 1:2000; 11,583,816,001) from Roche; mouse IgG isotype control (14–4732-82) was from Invitrogen; Alexa Fluor 488-, 555-, 594- and 647-conjugated secondary antibodies were from Thermo Fisher (A21206, A31570, A11032 and A21447, respectively).

Techniques: Expressing, Plasmid Preparation, Control, Staining

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy

doi: 10.1080/15548627.2021.1874660

Figure Lengend Snippet: The SQSTM1/p62[E409A,K420R] mutant rescues the accumulation of HTTQ109-GFP aggregates induced by BPLF1. (A) HeLa cells were co-transfected with plasmids expressing HTTQ109-GFP, FLAG-BPLF1 and increasing amount of HA-SQSTM1/p62[E409A,K420R] and cell lysates collected after 48 h were analyzed in filter trap assays and immunoblotting. Representative immunoblots illustrating the capacity of HA-SQSTM1/p62[E409A,K420R] to rescue the accumulation of HTTQ109-GFP aggregates in a dose dependent manner. (B) Quantification of data presented in A; means ± SEM of three independent experiments. (C) Representative immunoblots illustrating the failure to rescue the accumulation of HTTQ109-GFP aggregates upon expression of SQSTM1/p62[K7A]. (D) Quantification of data presented in C; means ± SEM of three independent experiments. Statistical analysis was performed using Student t-test. *P ≤ 0.05

Article Snippet: Antibodies and their manufacturers were: rat anti-BPLF1 [ ] (WB 1:1600, 2E5 supernatant) was from Helmholtz Zentrum (2E5); mouse anti-FLAG clone M2 (WB 1:4000, IF 1:400; F1804) and rabbit anti-LC3B (WB 1:1000, IF 1:200; L7543) were from Sigma-Aldrich; goat anti-FLAG (1:400; ab1257) and rabbit anti-SQSTM1/p62 (IP 1–2 μg/mg of lysate; ab101266), rabbit anti-GFP (WB 1:2000, ab290) and rabbit IgG isotype control (ab172730) were from Abcam; mouse anti-SQSTM1/p62 (WB 1:1000 IF 1:200; 610,832) was from BD Biosciences; rabbit anti-SQSTM1/p62 (WB 1:1000, 8025S) was from Cell Signaling Technology; mouse anti-HA clone 12 CA5 (WB 1:2000; 11,583,816,001) from Roche; mouse IgG isotype control (14–4732-82) was from Invitrogen; Alexa Fluor 488-, 555-, 594- and 647-conjugated secondary antibodies were from Thermo Fisher (A21206, A31570, A11032 and A21447, respectively).

Techniques: Mutagenesis, Transfection, Expressing, Western Blot

Journal: Nature Communications

Article Title: The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD

doi: 10.1038/s41467-024-50500-z

Figure Lengend Snippet: a Western blot showing elevated levels of FKBP51 and Snap23 in AKT2 overexpressing ARPE19 cells, which were reduced upon simultaneous upregulation of SIRT5 or overexpression of an AKT2 inactive mutant (K14A/R25E). n (biological replicate) = 4. b Immunofluorescence assay showing association of LC3-positive autophagosomes (green) with lysosomes (Lamp1-positive; red) in control ARPE19 cells (arrows in b ), which was significantly reduced upon AKT2 overexpression. Scale bar = 50 μm. n (biological replicate) = 8. c RNAseq analysis from RPE cells of 4-month-old WT and Akt2 KI mice showing differential expression of several autophagy-related genes. n = 3. d Western blot showing reduced expression of autophagy mediators ATG9B and ULK1, as well as upregulation of the autophagosome marker p62/SQSTM1 in Akt2 KI RPE cells, relative to WT. n (biological replicate) = 4. e Chromatin immunoprecipitation showing diminished binding of TFEB on Atg9b promoter region in Akt2 KI RPE cells, compared to WT. n (biological replicate) = 4. f Western blot showing reduced autophagy flux (Ratio of LC3-II/ Vinculin in BafA1 treated vs untreated) in Akt2 KI RPE explants compared to WT when treated with Bafilomycin A1 (BafA1; 1 μm) for 4 h. n (biological replicate) = 4. g ARPE19 cells were transfected with AKT2 construct for 48 h or left untreated (control), followed by an overnight infection with an Adenovirus-GFP-RFP-LC3B construct to label the autophagosomes (yellow) and autolysosomes (red). The number of autolysosomes (red puncta) was significantly decreased in AKT2 overexpressing cells (arrows in c ) when compared to controls, suggesting a decline in autophagy flux. Scale bar = 50 μm. n (biological replicate) = 4. h Transmission electron micrographs showing double membranous autophagosomes in the RPE cells of 15-month-old Akt2 KI mice, but not in age-matched WT. Scale bar = 600 nm. n = 5. All values are Mean ± S.D. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. The statistical tests used in ( a , c, and f ) is One-way ANOVA followed by Tukey’s post-hoc test whereas in ( b , d , g ) is Student’s t-test (Two-sided). The exact p-values are a FKBP51: P = 0.0007 (AKT2-GFP-pcDNA vs untr a nsfected), P = 0.0327 (AKT2-GFP-pcDNA+SIRT-HA-pcDNA vs AKT2-GFP-pcDNA), P = 0.0081 (AKT2(K14A/R25E)-GFP-pcDNA vs AKT2-GFP-pcDNA); b P = 1.598E-10 (AKT2-pcDNA vs Control); d ATG9B: P = 0.0139, p62: P = 0.0013, ULK1: P = 0.017; e P = 0.001 ( Akt2 KI vs WT); f P = 9.45202E-05 ( Akt2 KI vs WT); g Autophagosome: P = 0.0012, Autolysosome: P = 0.000082 (AKT2-pcDNA vs Control). Source Data is provided in the Source Data file.

Article Snippet: CTSL (NB100-1775), p62/SQSTM1 (NBP1-42821), and PGC-1α (NBP1-04676) were purchased from Novus Biologicals.

Techniques: Western Blot, Over Expression, Mutagenesis, Immunofluorescence, Control, Quantitative Proteomics, Expressing, Marker, Chromatin Immunoprecipitation, Binding Assay, Transfection, Construct, Infection, Transmission Assay

Journal: Nature Communications

Article Title: The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD

doi: 10.1038/s41467-024-50500-z

Figure Lengend Snippet: RPE cells from 6-month-old Cryba1 cKO mice treated with trehalose for 3 consecutive months show significant rescue in the levels of a CLEAR network genes like Ctsd , Lamp1 and Atp6v0a1 , b cathepsin D (CTSD) activity and c protein levels of the autophagosome marker p62/SQSTM1, relative to RPE cells from vehicle (water) treated Cryba1 cKO mice. n (biological replicate) = 4. RPE cells from 6-month-old Akt2 KI mice treated with trehalose for 3 consecutive months showed statistically significant rescue of d protein levels of autophagosome marker p62/SQSTM1 and lysosomal hydrolase, CTSD as well as e CLEAR network gene ( Ctsd , Ctsb , Lamp1 , Atp6v0a1 ) expression, compared to vehicle-treated Akt2 KI mice. n (biological replicate) = 4. Trehalose treatment to Akt2 KI mice showed noticeable rescue in f CTSD and g CTSL activities relative to untreated Akt2 KI mice. n (biological replicate) = 4. Hematoxylin-eosin stained sections and quantification showing a decrease in ONL thickness in h Akt2 KI and i Cryba1 cKO mice, which was rescued upon trehalose treatment. Prevalence of a patchy RPE layer with the accumulation of drusen-like deposits (asterisks in i and inset) in Cryba1 cKO retina (vehicle control), with rescue by trehalose treatment. Scale bar = 100 μm (inset = 20 μm). n (biological replicate) = 5. j Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en . Cartoon showing that AKT2 upregulation in RPE cells from Akt2 KI mice and CFH (H/H) iPSC-derived cells trigger deregulation of SIRT5/PGC-1α dependent lysosomal function. Trehalose and S467A TFEB mutant construct rescued the lysosomal abnormalities and delayed the progression of early RPE changes in a mouse model. All values are Mean ± S.D. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. The statistical test used in ( a – i ) is One-way ANOVA followed by Tukey’s post-hoc test. The exact p-values are a Ctsd : P = 0.0042, Lamp1 : P = 0.0010, Atp6v0a1 : P = 0.0062 ( Cryba1 cKO vs Cryba1 fl/fl ), Ctsd : P = 0.0129, Lamp1 : P = 0.0055, Atp6v0a1 : P = 0.0489 ( Cryba1 cKO+Trehalose vs Cryba1 cKO); b P = 0.00006 ( Cryba1 cKO vs Cryba1 fl/fl ), P = 0.0013 ( Cryba1 cKO+Trehalose vs Cryba1 cKO); c P = 0.0013 ( Cryba1 cKO vs Cryba1 fl/fl ), P = 0.0258 ( Cryba1 cKO+Trehalose vs Cryba1 cKO); d p62: P = 0.0037 and CTSD: P = 0.0461 ( Akt2 KI vs WT), p62: P = 0.0072 and CTSD: P = 0.0092 ( Akt2 KI+Trehalose vs Akt2 KI); e Ctsd : P = 0.00001, Ctsb : P = 0.00002, Lamp1 : P = 0.000035, Atp6v0a1 : P = 0.000029 ( Akt2 KI vs WT), Ctsd : P = 0.0020, Ctsb : P = 0.0005, Lamp1 : P = 0.0017, Atp6v0a1 : P = 0.0056 ( Akt2 KI+Trehalose vs Akt2 KI); f P = 0.00002 ( Akt2 KI vs WT), P = 0.000042 ( Akt2 KI+Trehalose vs Akt2 KI); g P = 0.000049 ( Akt2 KI vs WT), P = 0.00006 ( Akt2 KI+Trehalose vs Akt2 KI). h P = 0.00001 ( Akt2 KI vs WT), P = 0.0001 ( Akt2 KI+Trehalose vs Akt2 KI); i ONL thickness: P = 0.000027 ( Cryba1 cKO vs Cryba1 fl/fl ), P = 0.000019 ( Cryba1 cKO +Trehalose vs Cryba1 cKO), drusen-like deposits: P = 0.0005 ( Cryba1 cKO+Trehalose vs Cryba1 cKO; Student’s t-test). Source Data is provided in the Source Data file.

Article Snippet: CTSL (NB100-1775), p62/SQSTM1 (NBP1-42821), and PGC-1α (NBP1-04676) were purchased from Novus Biologicals.

Techniques: Activity Assay, Marker, Expressing, Staining, Control, Derivative Assay, Mutagenesis, Construct

Journal: Oncotarget

Article Title: Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

doi: 10.18632/oncotarget.15336

Figure Lengend Snippet: A . Cytotoxicity of Tan I in the H28 and H2452 mesothelioma cell lines. Two mesothelioma cell lines were treated with various concentrations of Tan I (0, 5, 10, 20, and 40 μM) for 24 h. Cell viability was determined by the MTT assay. B . The inhibitory effect of Tan I on colony formation in H28, and H2452 mesothelioma cells. The colony formation assay was performed on mesothelioma cells treated with Tan I (20 μM). C . Effect of Tan I on autophagic vacuoles in H28 cells by TEM observation. H28 cells were treated with Tan I (20 μM) for 24 h, and autophagic morphology was observed by TEM. The black arrow indicates an autolysosome that contains the remnants of digested organelles. High-magnification image of the black square (bottom panel). D . Effect of Tan I on acidic autophagic vacuoles in Tan I-treated H28 cells. H28 cells were treated with Tan I for 24 h, stained with acridine orange (AO) and observed under the FLUOVIEW FV10i confocal microscopy (Olympus, Tokyo, Japan). E . Effect of Tan I (20 μM) on punctae formation of endogenous LC3II in H28 and H2452 cells by immunofluorescence. Bar scale = 10 μM, DAPI-blue, endogenous LC3-Green. F . Effect of Tan I on PARP, p62 and LC3II in H28 and H2452 mesothelioma cells. G . Effect of Tan I on the mRNA expression of p62 and LC3II in H28 cells by RT-qPCR. Total RNA was isolated from Tan 1 treated H28 cells and RT-PCR was performed as shown in Materials and Methods.

Article Snippet: The lysates were pre-cleared with A/G agarose bead (Santa Cruz Biotechnology, sc-2003) and then immunoprecipitated at 4 °C with antibodies directed against p62 (Cell signaling, 7695) and rabbit IgG (Santa Cruz biotechnology, sc2313).

Techniques: MTT Assay, Colony Assay, Staining, Confocal Microscopy, Immunofluorescence, Expressing, Quantitative RT-PCR, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

doi: 10.18632/oncotarget.15336

Figure Lengend Snippet: A . Effect of Tan I on the colocalization of accumulated p62 with GFP-LC3II in H28 cells. B . The p62 ΔUBA domain is responsible for LC3II punctae formation by Tan I in H28 cells by immunofluorescence. H28 cells were transiently co-transfected with the Myc-p62 WT , Myc-p62 ΔUBA and Myc-p62 ΔLIR domain plasmids. The colocalization of LC3 and aggregates of full-length p62 or its deleted domain p62 plasmids was indicated by arrows. Bar scale =10 μm, GFP-LC3-Green, DAPI-blue, Myc-tagged p62-Red. C . Effect of Tan I on the expression of LC3II and p62 in H28 cells transfected with the Myc-p62 WT , Myc-p62 ΔUBA and Myc-p62 ΔLIR domain plasmids. Myc-tagged constructs of wild-type (WT) p62, p62 ΔUBA , and p62 ΔLIR were transfected into H28 cells in the presence or absence of Tan I, and Western blotting was performed with antibodies targeting p62, LC3II and β-Actin. An asterisk (*) indicates the p62 expression level of the Myc-tagged p62 constructs.

Article Snippet: The lysates were pre-cleared with A/G agarose bead (Santa Cruz Biotechnology, sc-2003) and then immunoprecipitated at 4 °C with antibodies directed against p62 (Cell signaling, 7695) and rabbit IgG (Santa Cruz biotechnology, sc2313).

Techniques: Immunofluorescence, Transfection, Expressing, Construct, Western Blot

Journal: Oncotarget

Article Title: Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

doi: 10.18632/oncotarget.15336

Figure Lengend Snippet: A . Effect of Tan I on ER stress-related proteins such as GRP78, CHOP, IRE1 and PERK in two mesothelioma cell lines. Western blotting was performed to determine the effect of Tan I on the expression of GRP78, CHOP, PERK, ATF4, ATF6, IRE1 and β-Actin in H28 and H2452 cells. B . Time-dependent effect of Tan I on IRE1 and CHOP expression in H28 cells. C . Effect of Tan I on the phosphorylation of JNK in H28 and H2452 cells. Two mesothelioma cell lines were exposed to Tan I and Western blotting was performed to determine the effect of Tan I on JNK phosphorylation. D . Effect of the JNK inhibitor SP600125 on the expression of p62, LC3II and IRE1 in H28 cells. H28 cells were treated with Tan I (20 μM) in the presence or absence of SP600125 for 24 h. Western blotting was performed to determine the levels of LC3 conversion, and p62/SQSTM1, IRE1 and β-Actin expression.

Article Snippet: The lysates were pre-cleared with A/G agarose bead (Santa Cruz Biotechnology, sc-2003) and then immunoprecipitated at 4 °C with antibodies directed against p62 (Cell signaling, 7695) and rabbit IgG (Santa Cruz biotechnology, sc2313).

Techniques: Western Blot, Expressing, Phospho-proteomics

Journal: Oncotarget

Article Title: Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

doi: 10.18632/oncotarget.15336

Figure Lengend Snippet: A . Effect of IRE-1 depletion by siRNA transfection on the ability of Tan I to activate p62 and LC3II in H28 cells. H28 cells were transfected with control or IRE-1 siRNAs. One day after transfection, H28 cells were treated with Tan I and Western blotting was performed to determine the levels of IRE-1, p62/SQSTM1, LC3 and β-Actin. The bar graph represents the densitometric analysis of p62. B . Binding of IRE1 and p62 in Tan I-treated H28 cells by the immunoprecipitation (IP) method. H28 cells were treated in the presence or absence of Tan I for 24 h. Endogenous p62/SQSTM1 was immunoprecipitated with a p62 antibody, and Western blotting was performed to determine the binding of IRE1 and p62/SQSTM1. C . Depletion of IRE1 or ATG5, but not p62 or Beclin-1, increases cytotoxicity in Tan I treated H28 cells. D . Effect of ATG5 depletion by siRNA on PARP, Procaspase 3 and LC3 II by Tan I treatment in H28 cells. E . Colocalization of Myc-tagged p62 with endogenous IRE1 was observed by immunofluorescence in Tan I-treated H28 cells. F . Protein-protein interactions between ATG5, JNK, IRE1, LC3II and CHOP shown in GeneCards database.

Article Snippet: The lysates were pre-cleared with A/G agarose bead (Santa Cruz Biotechnology, sc-2003) and then immunoprecipitated at 4 °C with antibodies directed against p62 (Cell signaling, 7695) and rabbit IgG (Santa Cruz biotechnology, sc2313).

Techniques: Transfection, Control, Western Blot, Binding Assay, Immunoprecipitation, Immunofluorescence, Protein-Protein interactions

Journal: Oncotarget

Article Title: Activation of JNK and IRE1 is critically involved in tanshinone I-induced p62 dependent autophagy in malignant pleural mesothelioma cells: implication of p62 UBA domain

doi: 10.18632/oncotarget.15336

Figure Lengend Snippet:

Article Snippet: The lysates were pre-cleared with A/G agarose bead (Santa Cruz Biotechnology, sc-2003) and then immunoprecipitated at 4 °C with antibodies directed against p62 (Cell signaling, 7695) and rabbit IgG (Santa Cruz biotechnology, sc2313).

Techniques: