ulk1  (Santa Cruz Biotechnology)

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: ( A ) Schematic of primary and validation screens. Readout is based on quantification of red mKeima puncta per cell. Representative images of siControl and siULK1 demonstrate image segmentation and feature collection of perinuclear mKeima puncta from acid pH (excitation ~586 nm). ( B ) Screening results plotted as log 2 of the mean FC of the three siRNAs for each gene relative to the negative control against the −log 10 of the combined P value. Selected candidates (RSA analysis) are annotated in red. ( C ) Venn diagram depicts overlap of significant hits from both cell lines of the validation screen with the FC criteria <0.5. ( D ) Lysosomal mKeima puncta for 19 candidates fulfilling the selection criteria from (C). Data are depicted as the mean of two siRNAs from two independent repetitions. Data normalized to positive (ULK1) and negative (Control) control siRNAs. ( E and F ) Representative images (60×) of Tig3 cells (72-hour transfection) in full medium (FM) or starved in HBSS for 2 hours, immunostained for LC3B (E) or ATG16L1 (F). Nuclear stain with Hoechst 33342. Magnified inserts of highlighted areas on the right. Scale bar, 5 μm. The number of puncta was quantified from ≥100 cells, and the data represent mean ± SD number of puncta per cell. ATG16L1 ( n = 2) and LC3B ( n = 3). **** P < 0.0001. ( G ) TEM analysis of Tig3 cells (72-hour transfection) starved for 2 hours (HBSS) in the presence of 200 nM BafA1 before fixation. Representative images are shown. Scale bar, 1 μm. Images displayed on the right are cropped and magnified from the highlighted area. Red arrows denote autophagic vesicles. Quantification of mean number of autophagic vesicles ± SD per cross section ( n ≥ 25 cross sections). Statistical analysis [(E) to (G)] was performed by unpaired Student’s t test. ** P < 0.01, **** P < 0.0001.

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Negative Control, Selection, Control, Transfection, Staining

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: ( A and C ) Tig3 cells (72-hour transfection) were starved for indicated intervals in HBSS (A) or starved for 16 hours before treatment with cycloheximide (CHX) (100 ng/ml) for indicated intervals (C). A representative Western blot is shown ( n = 3). ( B and D ) Densiometric quantifications relating to (A) and (C). Data represent relative ULK1 expression normalized to time point zero and total loading and are depicted as the mean ± SEM from three independent experiments. Statistical analysis was performed by two-way ANOVA. * P < 0.05 (B) and P = 0.0685 (D). ( E ) Western blot of liver tissue lysates from starved WT and KO mice. A representative blot from three mice of each genotype is shown. [(A), (C), and (E)] Gel stain is used as a loading control. ( F ) Densiometric quantification relating to (E). The graph depicts relative expression of ULK1 normalized to total loading. The data represent mean ± SD ( n = 6). Statistical analysis was performed by unpaired Student’s t test. ** P < 0.01. ( G ) Representative images from immunohistochemistry of starved mouse livers stained for ULK1. Scale bar 100 μm. ( H ) Representative images (60×) of WT and Peli3 KO cells starved for 2 hours in HBSS and immunostained for ULK1. Nuclear stain with Hoechst 33342. Magnified inserts shown on the right. Scale bar, 5 μm. ( I ) Quantifications relating to (H). ULK1 puncta quantified from ≥120 cells. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed by unpaired Student’s t test. **** P < 0.0001. ( J ) Representative Western blot of primary hepatocytes from Peli3 WT and KO mice. Starvation and BafA1 treatments were for 4 hours ( n = 3). ( K ) Densiometric quantification of phospho-ATG14 (P-ATG14) (Ser 29 ) and total ATG14 (T-ATG14) relating to (J). Data depict the mean ± SD from four independent experiments. Statistical analyses by one-way ANOVA. * P < 0.05.

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Transfection, Western Blot, Expressing, Staining, Control, Immunohistochemistry

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: ( A ) Co-IP from Tig3 cells expressing GFP or GFP-PELI3 in full medium or starved (HBSS, 2 hours). Western blot of inputs and IP fractions are shown from a representative experiment ( n = 3). ( B and C ) Top: Schematic of ULK1 (B) or PELI3 (C) domain organization and relevant deletion constructs. Bottom: Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( D ) Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). L.E, low exposure; H.E, high exposure. ( E ) Left: Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). Right: Corresponding densiometric quantification of ubiquitin levels normalized to bait. The data represent mean ± SD ( n = 3). Statistical analysis performed by unpaired Student’s t test. ** P < 0.01. ( F ) Co-IP from HEK293T cells expressing indicated constructs and treated with BafA1 or MG132 as indicated (2 hours). Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( G ) Co-IP of endogenous ULK1 from starved WT and Peli3 KO mouse liver tissue. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( H ) Left: Co-IP of endogenous ULK1 from WT and Peli3 KO mouse-derived primary hepatocytes in full medium or starved. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). Right: Corresponding densiometric quantification of ubiquitin levels normalized to bait from starved samples. The data represent mean ± SD ( n = 3). Statistical analysis performed by unpaired Student’s t test. * P < 0.05.

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Co-Immunoprecipitation Assay, Expressing, Western Blot, Construct, Derivative Assay

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: In response to nutrient deprivation, PELI3 safeguards ULK1 homeostasis through ubiquitination of ULK1. This is facilitated by PELI3’s interaction with ATG8 proteins. In the absence of PELI3, ULK1 accumulates, leading to a disruption of autophagy and a decreased degradation of LDs in the liver, causing enhanced hepatic steatosis. Created with BioRender (Frankel, 2023; https://BioRender.com/a71f180 ).

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Disruption