Journal: Life Science Alliance

Article Title: Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity

doi: 10.26508/lsa.202000815

Figure Lengend Snippet: (A) MEF cells expressing GFP-LC3 and mCherry-DFCP1 were pretreated for 15 min with 1 μM MRT68921 or DMSO followed by treatment with EBSS or EBSS and 1 μM MRT69821. The cells were then immediately subjected to live imaging using a Nikon Eclipse Ti wide-field microscope. (B) Representative images of cells are shown at 30 min in and asterisk marks the section highlighted in panel (B). The movies are provided as supplementary information ( and ). Scale bar, 10 μm. (A, B) Montage of the marked areas from the movies shown in (A) from minutes 22–41. Arrowheads indicate colocalisation between GFP-LC3 and mCherry-DFCP1. (B, C) Graphical representation of the maximum intensity of the mCherry-DFCP1 (red) and GFP-LC3 (green) puncta indicated with arrows in (B). (D) Quantitation of the mean time for which GFP-LC3 and mCherry-DFCP1 signals colocalised on the same punctate structure (from n = 4 events, bars represent mean with SEM). (E) Immunoblot of WT MEF cells treated with EBSS and 1 μM MRT68921 as indicated. (F) Quantitation of (E). pS318-ATG13 levels were normalised to total ATG13 (left) and LC3-II levels were normalised to Tubulin (right). Data represent mean of n = 4 ± SEM. Statistical analysis was performed with two-way ANOVA and a Tukey’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, **** = P < 0.0001 and ns, not significant. Source data are available for this figure.

Article Snippet: The following antibodies were purchased from CST: ULK1 (8054S), pS757 ULK1 (6888T), pS555 ULK1 (5869S) and LC3 A/B (4108S—used for Western blot). pS318-ATG13 (NBP2-19127) was from Novus Biologicals.

Techniques: Expressing, Imaging, Microscopy, Quantitation Assay, Western Blot

Journal: Life Science Alliance

Article Title: Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity

doi: 10.26508/lsa.202000815

Figure Lengend Snippet: (A) Immunoblot of lysates from MEF cells treated with EBSS, 50 nM BafA1, 1 μM MRT68921, 10 μM SBI0206965, and 1 μM ULK-101 for 1 h as indicated. (B) Quantitation of pS318-ATG13 levels normalised to total ATG13 (top) and LC3-II levels normalised to α-Tubulin (bottom). Data represent mean of n = 4 ± SEM. Statistical analysis was performed with two-way ANOVA and a Tukey’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001 and ns, nonsignificant (unless indicated comparisons of each treatment are with the relevant DMSO control). (C) Representative flow cytometry assay of MEF cells expressing mCherry-GFP-LC3 treated with either DMSO or 50 nM BafA1, or 4 μM MRT68921, or 20 μM SBI0206965, or 2 μM ULK-101 in EBSS for 4 h as indicated, where a decrease/increase in GFP and mCherry expression can be observed and quantified to measure the percent of cells undergoing autophagy. The number in each panel indicates % of cells undergoing autophagy. Quantitation of the flow data is shown on the right and represents mean of n = 3 ± SEM. Statistical analysis was performed with one-way ANOVA and a Tukey’s multiple comparisons test. **** = P < 0.0001. Source data are available for this figure.

Article Snippet: The following antibodies were purchased from CST: ULK1 (8054S), pS757 ULK1 (6888T), pS555 ULK1 (5869S) and LC3 A/B (4108S—used for Western blot). pS318-ATG13 (NBP2-19127) was from Novus Biologicals.

Techniques: Western Blot, Quantitation Assay, Control, Flow Cytometry, Expressing

Journal: Life Science Alliance

Article Title: Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity

doi: 10.26508/lsa.202000815

Figure Lengend Snippet: (A, B) Quantification of pS757 ULK1 levels (A) and pS555 ULK1 levels (B) normalised to total ULK1. Bars represent mean of n = 4 ± SEM. Statistical analysis was performed with a two-way ANOVA and a Tukey’s multiple comparisons test. The presence of the inhibitors resulted in no significant changes among the different conditions. This figure is complementary to . (C) Co-immunoprecipitation of ULK1 with FIP200 and ATG13 from cells growing in Fed or EBSS conditions treated with 2 μM MRT68921, or 1 μM ULK-101, or 5 μM SBI0206965 or DMSO for 1 h, as indicated. Source data are available for this figure.

Article Snippet: The following antibodies were purchased from CST: ULK1 (8054S), pS757 ULK1 (6888T), pS555 ULK1 (5869S) and LC3 A/B (4108S—used for Western blot). pS318-ATG13 (NBP2-19127) was from Novus Biologicals.

Techniques: Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Necroptosis as a Novel Facet of Mitotic Catastrophe

doi: 10.3390/ijms23073733

Figure Lengend Snippet: Crosstalk between necroptosis, autophagy, and apoptosis after mitotic catastrophe induction. ( A ) Caov4 and HCT116 cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 for 48 h. Immunoblot analysis using the indicated antibodies is shown. ( B ) Sub-G1 analysis of Caov4 and HCT116 cells treated with 600 nM doxorubicin and 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 for 48 h. Values are the mean (±standard deviation of the mean) of three independent experiments. ( C ) U1810 wild type and ATG13-knockout cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk and/or 30 μM necrostatin-1s for 24 h. Immunoblot analysis using the indicated antibodies is shown. * p < 0.05, ** p < 0.01.

Article Snippet: The membranes were incubated with primary antibodies against cleaved PARP-1 (Asp214) (9541S), RIP1 (4926S), phospho-RIP (Ser166) (65746S), caspase-3 (9662S), cleaved caspase-3 (9664S), and ATG13 (6940S) (all from Cell Signaling Technology, Danvers, MA, USA), PARP-1 (ab137653), p62/SQSTM1 (ab56416), LC3B (ab51520), and vinculin (ab129002) (all from Abcam, Eugene, OR, USA), and FADD (gift from Prof. Inna Lavrik, Otto von Guericke University Magdeburg, Magdeburg, Germany) overnight at 4 °C.

Techniques: Western Blot, Standard Deviation, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Necroptosis as a Novel Facet of Mitotic Catastrophe

doi: 10.3390/ijms23073733

Figure Lengend Snippet: Caspase inhibition increases the population of cells with mitotic catastrophe morphology. ( A ) Quantification of mitotic catastrophe after 48 h treatment with 600 nM doxorubicin in combination with 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 in Caov4 and HCT116 cells. ( B ) Quantification of mitotic catastrophe after 24 h of treatment with 600 nM doxorubicin in combination with 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s in wild type and ATG13-knockout U1810 cells. The number of mitotic catastrophe cells examined in each cell line are shown in the bars. Values are the mean (±standard deviation of the mean) of three independent experiments. * p < 0.05, ** p < 0.01, ns—not significant.

Article Snippet: The membranes were incubated with primary antibodies against cleaved PARP-1 (Asp214) (9541S), RIP1 (4926S), phospho-RIP (Ser166) (65746S), caspase-3 (9662S), cleaved caspase-3 (9664S), and ATG13 (6940S) (all from Cell Signaling Technology, Danvers, MA, USA), PARP-1 (ab137653), p62/SQSTM1 (ab56416), LC3B (ab51520), and vinculin (ab129002) (all from Abcam, Eugene, OR, USA), and FADD (gift from Prof. Inna Lavrik, Otto von Guericke University Magdeburg, Magdeburg, Germany) overnight at 4 °C.

Techniques: Inhibition, Knock-Out, Standard Deviation

Journal: Molecular Cell

Article Title: The Cargo Receptor NDP52 Initiates Selective Autophagy by Recruiting the ULK Complex to Cytosol-Invading Bacteria

doi: 10.1016/j.molcel.2019.01.041

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-ATG13 (E1Y9V) , Cell Signaling Tehnology , Cat# 13468.

Techniques: Luciferase, Transduction, Recombinant, Plasmid Preparation, Protease Inhibitor, Negative Control, Software, Chromatography, Molecular Weight

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-ATG13 , Sigma-Aldrich , Cat# SAB4200100; RRID:AB_10602787.

Techniques: Recombinant, Western Blot, Software

Journal: eLife

Article Title: Inhibition of ULK1/2 and KRAS G12C controls tumor growth in preclinical models of lung cancer

doi: 10.7554/eLife.96992

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-ATG13 (rabbit monoclonal) , Cell Signaling Technology , Cat# 13272 , ELISA (1:500).

Techniques: Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Journal of Virology

Article Title: Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

doi: 10.1128/JVI.01537-21

Figure Lengend Snippet: Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Antibodies against Atg5 (2630), p-AKT (4060), AKT (4685), p-mTOR (5536), mTOR (2983), p-S6 (4858), S6 (2317), p-4E-BP1 (2855), 4E-BP1 (9644), p-AMPK (2535), AMPK (5831), p-TSC-2 (3614), TSC-2 (4308), p-Raptor (2083), Raptor (2280), p-Atg13 (26839), Atg13 (13273), ULK1 (8054), p-ULK1 (Ser757) (14202), p-ULK1 (Ser555) (5869), VPS15 (14580), VPS34 (4263), Beclin1 (3495), p-Beclin1 (84966), Atg14 (96752), Atg16L1 (8089), p62 (16177), and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Virus, Infection, Western Blot, Membrane, Quantitative RT-PCR, Knockdown, Transfection, Control