Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 2. Inhibition of ULK1 results in a decrease of pICln phosphorylation in vivo A, Colocalization of ULK1 and pICln phospho and total. Cells were fixed with 4% PFA and afterwards permeabilized with Triton X-100 to visualize ULK1 (red) and pICln total or phosphospecific pICln (green). The DNA was stained with DAPI (blue), scale bars 10 µm. B, HEK293T cells were treated with 30 µM ULK inhibitor MRT67307 for 5 h or transfected with 50 nM Non-targeting control pool (NT) control or ULK1 siRNA for 72 h. Cells were fixed as described in A to visualize ULK1 (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. C, HEK293T cells were treated as described in B to visualize phosphospecific pICln (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. D, Inhibition or knockdown of ULK1 decreases pICln phosphorylation in vivo. Quantification of the intensity ratio between phosphospecific pICln and pICln total was made in Fiji and the diagram in Origin, the values for MRT67307 treatment are normalized on HEK wt value, and the values for ULK1 siRNA are normalized on NT control. The significance of the values was analyzed using a Mann-Whitney U test, the p-value of HEK wt vs. MRT67307 and NT control vs. ULK1 siRNA was below 0.005, indicating significant differences between the values.

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Inhibition, Phospho-proteomics, In Vivo, Staining, Transfection, Control, Knockdown, MANN-WHITNEY

Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 4. ULK1 phosphorylation of pICln increases UsnRNP biogenesis and spliceosomal activity A, The HIV-1-based splicing reporter contains a test 5‘splice site (5‘ss) with a mutated version of viral 5′ss D4 termed „− 1G3U“. This splice site carries two nucleotide substitutions: A-to-G at position − 1 (−1 G) and A-to-U at position + 3 (+3 U) and is poorly recognized by the endogenous U1 snRNA due to a mismatch at position + 3. However, splice site recognition is efficiently restored following the co-expression of a U1 snRNA with a compensatory U-to-A nucleotide exchange at position + 6, indicating that the modified U1 is assembled into functional snRNP particles within the cell. Uppercase letters within the splice site sequences represent complementary residues, while lowercase letters represent mismatches to the U1. Mutations are highlighted in red. Base-pairing at position + 3 is highlighted by a light red box and increased font size. B; C, HEK293T cells were treated with 30 µM inhibitor MRT67307 and transiently transfected with different splicing reporter constructs (see methods section for more details). After 20 h of inhibitor treatment cells were harvested to perform RNA isolation. Quantitative RT-PCR was executed and mRNA was analyzed by assessing the respective ratio of spliced/unspliced form, the p value was below 0.005 (B). The equal averages including differences between inhibitor- treated and untreated spliced and unspliced forms are listed in C (n = 3).

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Phospho-proteomics, Activity Assay, Expressing, Modification, Functional Assay, Transfection, Construct, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: NAD + augmentation restores mitophagy and limits accelerated aging in Werner syndrome

doi: 10.1038/s41467-019-13172-8

Figure Lengend Snippet: NAD + ameliorates premature aging in WS through DCT-1 and ULK1-dependent mitophagy. a Mitochondrial content was evaluated by quantifying MitoTracker green pixels of stained N2 and wrn-1 (gk99) worms at adult D1 and D7 ( n = 80 worms; Two-way ANOVA). values pooled from three independent biological repeats. Data from Vehicle-treated worms are also used in Fig. . b Quantified scores of muscle mitochondrial morphology of adult D7 N2 and wrn-1 ( gk99 ) worms. A myo-3::gfp reporter gene was expressed in body wall muscles. ( n = 20 worms/group; Two-way ANOVA). c , d Relative mitophagy rate in adult D7 N2 and wrn-1 ( gk99 ) worms. Mitophagy events were calculated as the co-localization between DsRed::LGG-1 and DCT-1::GFP in muscle cells. ( n = 20 worms for each condition; Two-way ANOVA). e Effects of NR or UA treatment on pharyngeal pumping in N2 and the wrn-1 ( gk99 ) worms at adult D4 and D6. ( n = 3 biologically independent experiments with 10–20 worms for each condition; Two-way ANOVA). f Pharyngeal pumping rates in adult D7 worms of designated groups. ( n = 20 worms/group; Two-way ANOVA). g , h mRNA levels of dct-1 ( d ) and unc-51 ( e ) in adult D7 worms. ( n = 3 biologically independent experiments; Two-way ANOVA). i Flow cytometry quantification of relative mitophagy incidence in HT01, WS01, and WRN-KD cells under different conditions. For siRNA control, siRNA-vector was added cells. ( n = 3 biologically independent experiments; Two-way ANOVA). j Western blot data showing changes of expression of designated proteins. Source data are provided as a Source Data file. Two-way ANOVA followed by Tukey’s post-hoc tests. Data are shown in mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For the ULK1 and AMPK knockdown in human cells, we use ULK1 siRNA (Origene, #SR322391) and AMPK siRNA (Origene, #SR321409) transient transfection in WS01 fibroblasts.

Techniques: Staining, Flow Cytometry, Plasmid Preparation, Western Blot, Expressing

Journal: Nature Communications

Article Title: NAD + augmentation restores mitophagy and limits accelerated aging in Werner syndrome

doi: 10.1038/s41467-019-13172-8

Figure Lengend Snippet: Effects of PARP1, sirtuins on the healthspan, and of NR on DNA repair in the wrn-1 worms. a Pumping rate of the adult D6 worms. Data are shown in mean ± S.E.M ( n = 20 worms per condition). For all the experiments, worms from L4 stage were exposed to vehicle control, the NAD + precursors NR and NMN (both at 1 mM), a SIRT1activator SRT1720 (10 µM), or a PARP inhibitor Olaparib (500 nM). Two-way ANOVA followed by Tukey’s post-hoc tests was used for data analysis. b Embryonic homologous recombination (HR) capacity was measured by scoring the percent survival of early stage embryos after irradiation with 90 Gy. Two HR mutants, brc-1(tm1145) and brd-1(dw1) , were used as positive controls, while two NHEJ mutants, cku-70(tm1524) and cku-80(ok861) , were negative controls . The results were from four biological replicates, including 400–900 worms for each condition. Data shown are mean ± S.E.M. (One-way ANOVA). c , d Changes of RAD-51 signals in the mitotic region of designated groups of worms. Worms were exposed to 90 Gy ionizing radiation and germlines were isolated 24 h post-irradiation. Immunostaining of DAPI and RAD51 in the germlines were performed using standard protocols (Two-way ANOVA was used for data analysis with * p < 0.05, ** p < 0.01, *** p < 0.001). For ( c ), scale bars, 10 µm. e Working model. We propose WRN mutation leads to cellular NAD + reduction through the down-regulation of the NAD + synthetic enzyme NMNAT1 and upregulation of cellular NAD + consumption (e.g., by PARPs). Cellular NAD + depletion impairs mitophagy through reduction of the activities of p-AMPK and p-ULK1, two upstream proteins which regulate autophagy/mitophagy. This results in accumulation of damaged mitochondria and impaired mitochondrial homeostasis. In combination with the defects in this NAD + -AMPK-ULK1-mitophagy pathway and other WRN-dependent cellular processes, such as DNA repair, they drive defective metabolism and accelerated aging. NAD + augmentation through NAD + precursors, such as NR (nicotinamide riboside) and NMN (nicotinamide mononucleotide) alleviate WRN mutation -induced pathological features in both C. elegans and Drosophila models of WS and in primary human cells from WS patients.

Article Snippet: For the ULK1 and AMPK knockdown in human cells, we use ULK1 siRNA (Origene, #SR322391) and AMPK siRNA (Origene, #SR321409) transient transfection in WS01 fibroblasts.

Techniques: Homologous Recombination, Irradiation, Isolation, Immunostaining, Mutagenesis

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots of total and phosphorylated ULK1, and total AMPKα and phospho-AMPKα Thr172 expression, in the kidneys of sham and CKD (5/6 nephrectomy) mice (n = 5). B. AMP/ATP ratio in the kidneys of WT mice in the sham and CKD groups (n = 5). C. Representative immunoblots of total AMPKα and phospho-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 5). D. AMP/ATP ratio in kidneys of WT or Ulk1 −/− mice (n = 5). E. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without 5-aminoimidazole 4-carboxamide-1-beta-d- ribofuranoside (AICAR) (1.0 mg/g BW) (n = 6). F. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P-ACC Ser79 , and P-RAPTOR Ser792 expression in liver tissue of WT or Ulk1 −/− mice treated with or without AICAR (n = 6). G. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 in white adipose tissue of WT or Ulk1 −/− mice treated with or without AICAR (n = 6). H. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without AICAR for 12 h (n = 3). P values (* P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Expressing, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing direct interaction between AMPKγ1 and ULK1. B. Evolutionally conserved sequence alignment of AMPKγ1 in mammals. C. Immunoblot of proteins subjected to a kinase assay using purified trimeric AMPK consisting of AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A mutant with and without ULK1 showing direct phosphorylation of AMPKγ1 at S260/T262 by ULK1. D. Immunoblots of proteins subjected to a kinase assay using purified monomeric AMPKγ1 and ULK1. E. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing AMPKγ1 bound to T-ULK1 is likely to be phosphorylated at S260/T262.

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Immunoprecipitation, Expressing, Sequencing, Western Blot, Kinase Assay, Purification, Mutagenesis

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 with/without Halo-tagged ULK1 expression in HEK293T cells showing AMPK bound to T-ULK1 is likely to be phosphorylated at P-AMPKα Thr172 . Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression when ULK1 is overexpressed (n = 3). A. B. Immunoprecipitation of FLAG-tagged AMPKγ1 WT or AMPKγ1 S260A/T262A with Halo- tagged ULK1 expression in HEK293T cells. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A when ULK1 is overexpressed (n = 4). B. Immunoprecipitation of FLAG-tagged AMPKγ1 with Halo-tagged ULK1 expression in HEK293T cells treated with/without BL918 (selective ULK1 activator) for 15 minutes. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 (lower) and P-AMPKα Thr172 (upper) expression (n = 3). C. Immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A with/without 15 minutes BL918 (selective ULK1 activator, 5 μM) treatment (n = 3). D. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with/without BL918 for 15 minutes (n = 3). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Immunoprecipitation, Expressing, Western Blot, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 8). B. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in livers of WT or Ulk1 −/− mice following starvation (n = 6). C. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 expression in kidneys of mice subjected to sham operation or 5/6 nephrectomy to induce chronic kidney disease (CKD) (n = 8). P values ( * P < 0.05, ** P < 0.01) were calculated using a two-tailed unpaired t -test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A, B. Representative immunoblots (A) and densitometric analysis (B) evaluating the sensitivity of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or S260A/T262A mutant to 12h-AICAR treatment (n = 3). A. C. Crystal structure revealing the role of Ser260 and Thr262 in maintaining the structure of AMPKγ1. B. D. Fluorescence intensity of Mant-AMP (fluorescent dye) bound to purified trimeric AMPK protein consisting of AMPKγ1 WT or AMPKγ1 S260A/T262A with/without ULK1 (n = 5) P values ( * P < 0.05) were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Mutagenesis, Fluorescence, Purification

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A treated with/without MK8722 (n = 3). B. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without MK8722 treatment (n = 3). C. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without MK8722 at 30 mg/kg BW (n = 5). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or a one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Expressing, Transfection, Control, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Mitochondrial ROS-derived PTEN oxidation activates PI3K pathway for mTOR-induced myogenic autophagy

doi: 10.1038/s41418-018-0165-9

Figure Lengend Snippet: Mitochondrial ROS-induced mTOR activation phosphorylates ULK1 at serine 317 for autophagy initiation and stimulates expression of autophagy-related proteins during muscle differentiation. a C2C12 cells were incubated in differentiation medium up to 5 days and were harvested every day. b Cells were incubated with 125, 250, or 500 nM MitoQ (MQ) or 125, 250, or 500 pM rapamycin for 5 days. MitoQ-containing or rapamycin-containing media were changed every day. c Cells were treated with 0.25 mM d-galactose with or without 0.015 U/ml galactose oxidase in DM for 5 days. Under these conditions, cells were treated with 250 nM MitoQ from 2 days after induction of differentiation and further incubated for 3 days. Cells were treated with 500 pM rapamycin in DM for 5 days. The levels of phospho-ULK1 at serine 317 (S317), serine 637 (S637) and serine 757 (S757), ULK1, phosphor-AMPK (p-AMPK (T172)), AMPK, phospho-ACC (p-ACC (S79)), ACC, Atg3, Atg5, Atg7, Atg12, Atg12–Atg5, LC3 (LC3-I, upper band; LC3-II, lower band), and MHC were assessed by Western blot analysis. β-Actin was used as a loading control. d Co-immunoprecipitation of mTOR (S2448) with phospho-ULK1 (S757 or S317) in cells treated without or with 250 nM MQ or 500 pM rapamycin. Expressions of mTOR and ULK1 were assessed by Western blot analysis. β-Actin was used as a loading control. e ULK1 phosphorylation. L6 or H9c2 cells were incubated in differentiation medium up to 5 days and were harvested every day. f Co-immunoprecipitation of mTOR (S2448) with phospho-ULK1 (S757 or S317) in L6 or H9c2 cells. g mTOR and ULK1 protein expressions in C2C12 and L6 cell lysates or mouse and rat tissue lysates were assessed by Western blot analysis. β-Actin was used as a loading control. C2 C2C12, B brain, H heart, Iʟ large intestine, Is small intestine, K kidney, Lu lung, Li liver, SM skeletal muscle, Sp spleen, and St stomach. h Cells were incubated in proliferation medium with or without 25 mM glucose for 24 g. Western blot analysis was conducted by specific antibodies using cell lysates. i Immunoprecipitation of phospho-ULK1 (S757) in C2C12, L6, or H9c2 cells. Western blot analysis was conducted by specific antibodies using cell lysates. β-Actin was used as a loading control. MQ MitoQ, Rapa or RP rapamycin, GA d-galactose, GO galactose oxidase

Article Snippet: siRNA for PTEN was acquired from Cell Signaling. siRNAs for mTOR and Raptor were obtained from Sigma-Aldrich. siRNAs for Rictor, ULK1, Atg5, Atg7, and Atg12 were purchased from Bioneer.

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Control, Immunoprecipitation, Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: Mitochondrial ROS-derived PTEN oxidation activates PI3K pathway for mTOR-induced myogenic autophagy

doi: 10.1038/s41418-018-0165-9

Figure Lengend Snippet: PTEN oxidation and mTOR activation are induced during muscle regeneration in mouse model. a Mouse left tibialis anterior (TA) muscles were injected with 50 μl (10 mM) cardiotoxin (CTX). Mice were sacrificed at indicated days after the injection (N = 5). b Mouse TA muscle tissues were homogenized and assessed by Western blot analysis. Oxidized and reduced PTEN proteins were resolved by non-reducing SDS-PAGE. The levels of phospho-PTEN (p-PTEN), PTEN, SOD2, and MHC proteins were assessed by Western blot analysis. c The levels of phospho-AKT at threonine 308 (T308) and serine 473 (S473), AKT, phospho-mTOR at serine 2448 (S2448) and serine 2481 (S2481), phospho-p70S6K at serine 371 (S371), threonine 389 (T389) and threonine 421/serine424 (T421/S424), p70S6K, phospho-4E-BP1 at threonine 37 and 46 (p-4E-BP1), and 4E-BP1 were determined by Western blot analysis. d The levels of phospho-ULK1 at serine 317 (S317), serine 637 (S637) and serine 757 (S757), ULK1, Atg3, Atg5, Atg7, Atg12, Atg12–Atg5, LC3 (LC3-I, upper band; LC3-II, lower band) were assessed by Western blot analysis. β-Actin was used as a loading control

Article Snippet: siRNA for PTEN was acquired from Cell Signaling. siRNAs for mTOR and Raptor were obtained from Sigma-Aldrich. siRNAs for Rictor, ULK1, Atg5, Atg7, and Atg12 were purchased from Bioneer.

Techniques: Activation Assay, Muscles, Injection, Western Blot, SDS Page, Control