Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: NBR1 enhances Smurf1-mediated p62 liquid-droplets accumulation. A , B LN229 cells were transfected with HA or HA-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-NDP52, anti-OPTN, anti-BAG3, anti-HA, and anti-β-actin) ( A ). Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin. Values were normalized against the intensity of LN229 transfected with HA; mean ± SD from 3 independent experiments. NS p > 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test ( B ). C , D LN229 cells transfected with RFP-Smurf1 and GFP-p62 were treated with control or NBR1 siRNA oligos, fixed after 24 h transfection with HA or HA-NBR1. The nucleus was stained by DAPI. Bar: 5 µm ( C ). Bar graphs indicate the ratio of the number of RFP-Smurf + GFP-p62 + puncta to the number of GFP-p62 + puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05 as determined by unpaired two-tailed Student’s t -test ( D ). E , F LN229 cells treated with control, NBR1, or Smurf1 siRNA oligos were transfected with GFP, GFP-Smurf1, or GFP-NBR1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 antibody. Bar: 5 µm. The nucleus was stained by DAPI ( E ). Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test ( F ). G LN229 cells overexpressing Flag-Smurf1 were transfected with GFP or GFP-NBR1, fixed after treating with BafA1 (100 nM, 24 h) and/or MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 antibody. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, * p < 0.05 as determined by unpaired two-tailed Student’s t -test. H , I LN229 cells transfected with RFP-GFP-p62 were treated by control, Smurf1 or NBR1 siRNA oligos, treated with MG132 (20 µM, 12 h), and fixed after transfection with HA, HA-NBR1 or Flag-Smurf1. The nucleus was stained by DAPI. Bar: 5 µm ( H ). Bar graphs indicate the number of RFP-GFP-p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t -test ( I ). J LN229 cells transfected with GFP-p62 and Flag-Smurf1 cultured in glass-bottom plates were treated with control or NBR1 siRNA oligos, then overexpressed HA or HA-NBR1. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. K LN229 cells transfected with GFP-p62 were treated with control or Smurf1 siRNA oligos, and overexpressed HA or HA-NBR1. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments

Article Snippet: Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

Techniques: Transfection, Western Blot, Two Tailed Test, Control, Staining, Immunofluorescence, Cell Culture

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: Smurf1 mediated NBR1 expression in p62 dependent manner. A LN229 cells treated with control, p62 or NBR1 siRNA oligos were transfected with Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-NBR1, anti-p62, and anti-β-actin). B 293T cells treated with control or p62 siRNA oligos were transfected with HA or HA-Smurf1, and immunoprecipitated by HA antibody. The immunoprecipitates and the input were probed with anti-p62, anti-NBR1, and anti-HA antibodies. C LN229 cells treated with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NBR1 mRNA. Values were normalized against the amount of LN229 transfected with si-Control and Flag; mean ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t -test. D LN229 cells treated with control or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-p62, anti-NBR1, anti-Smurf1 and anti-β-actin). E LN229 cells treated with control or Nrf2 siRNA oligos were transfected with Flag or Flag-Smurf1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-NBR1 and anti-p62 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of NBR1 + p62 + puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. F LN229 cells treated with control, Nrf2, or p62 siRNA oligos were transfected with HA or HA-NBR1, and then overexpressed Flag or Flag-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-p62 S349 , anti-p62, anti-Flag, anti-HA, anti-Nrf2, and anti-β-actin)

Article Snippet: Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

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

Journal: Cell & Bioscience

Article Title: Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

doi: 10.1186/s13578-023-00978-9

Figure Lengend Snippet: NBR1 enhances Smurf1-drived Nrf2-mediated oxidative stress response. A LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Smurf1, anti-NBR1, anti-p62, and anti-β-actin). B LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1 and NBR1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (mean ± SD from 3 independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. C LN229 cells were fixed after treating with control (PBS) or H 2 O 2 (200 µM, 2 h), and then immunofluorescence stained with anti-Smurf1, anti-p62, and anti-NBR1 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). ** p < 0.01 as determined by unpaired two-tailed Student’s t -test. D LN229 cells were transfected with control or Smurf1 siRNA oligos. Thereafter, the cells were divided into three groups: (i) cultured in regular medium, (ii) treated with H 2 O 2 (200 µM, 2 h), and (iii) after treating with H 2 O 2 (200 µM, 2 h), cultured in regular medium for another 12 h. Cell lysates were prepared and subjected to western blot analysis with indicated antibodies (anti-NBR1, anti-p62, anti-p-p62 S349 , anti-p-p62 S403 , anti-Smurf1, and anti-β-actin). E LN229 cells transfected with control or Smurf1 siRNA oligos were treated with control (PBS) or H 2 O 2 (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1, p62, NBR1, and NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (mean ± SD from 3 independent experiments). NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. F LN229 cells treated with control or Smurf1 siRNA oligos and fixed after treating with control (PBS) or H 2 O 2 (200 µM, 2 h), and then immunofluorescence stained with anti-NBR1, anti-Smurf1 and anti-p62 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. G LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H 2 O 2 (200 µM, 2 h) after overexpressing HA or HA-NBR1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-Flag, and anti-β-actin). H LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H 2 O 2 (200 µM, 2 h) after overexpressing HA or HA-NBR1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t -test. I Role of Smurf1 in stress response. Stress upregulates the level of Smurf1, leading to the increased formation and material exchange of p62 liquid droplets. Smurf1 promotes the phosphorylation of p62 S349 by activating mTORC1 signalling pathway. These further promote the transcription of anti-stress proteins by competitively binding Keap1 and mediating Nrf2 nuclear import. The activated Nrf2 increases the mRNA level of Smurf1, p62, and NBR1 to promote the formation and enlarging of p62 liquid droplets as positive feedback

Article Snippet: Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

Techniques: Transfection, Control, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Cell Culture, Phospho-proteomics, Binding Assay

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: (A) Cross-sections of quadriceps from control (Ulk1+/−Ulk2−/flox) and Ulk1/2 hypomorph (Ulk1−/− Ulk2−/flox) mice at 24 weeks were stained with H&E and modified Gomori trichrome staining. Arrow indicates a crescentic inclusion and arrowhead indicates a vacuole. Scale bars, 25 μm.

Article Snippet: The pMXs-IP-EGFP-ULK1 (38193) and pMXs-IP-EGFP-ULK2 (38201) were obtained from Addgene.

Techniques: Staining, Modification

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: (A) HEK293T cells were transfected with ULK1-DDK or ULK2-DDK and subjected to heat shock at 43°C for 1 h. Immediately following heat shock, cells were lysed and immunoprecipitated for DDK, followed by immunoblot against the indicated proteins.

Article Snippet: The pMXs-IP-EGFP-ULK1 (38193) and pMXs-IP-EGFP-ULK2 (38201) were obtained from Addgene.

Techniques: Transfection, Immunoprecipitation, Western Blot

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: The pMXs-IP-EGFP-ULK1 (38193) and pMXs-IP-EGFP-ULK2 (38201) were obtained from Addgene.

Techniques: Recombinant, Western Blot, Software

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , The amino-acid sequence of human MEK1 aligned to human ULK1 and human ULK2. Black letters, amino acids; blue letters, the four amino acids mutated in C u- b inding m utant (CBM) of MEK1 to decrease Cu binding and those conserved between ULK1 and ULK2. b , c , Immunoblot detection of recombinant GST-ULK1 or GST-ULK2 bound to a resin charged with or without Cu, Fe, or Zn compared to input. d , e , f , g , Immunoblot detection of recombinant phosphorylated (P)-ATG13, total (T)-ATG13, and GST-ULK1 or GST-ULK2 from ULK1 or ULK2 in vitro kinase assays treated with or without increasing concentrations of CuCl 2 from 0 to 10 μM ( d , e ), with or without increasing concentrations of TTM from 0 to 50 μM, or 10 μM MRT68921 ( f , g ). Quantification: ΔP-ATG13/T-ATG13 normalized to GST-ULK1 and GST-ATG13 alone. h , Immunoblot detection of P- and/or T-mTOR, p70S6K, ULK1, ATG13, CCS, or β-ACTIN from Ctr1 -/- MEFs stably expressing CTR1 WT (WT) or empty vector (VO) treated with vehicle (VEH) or amino acid deprivation (-AA). Quantification: ΔP-ATG13/T-ATG13 normalized to WT, VEH control. i , j , Immunoblot detection of recombinant P-ATG13, T-ATG13, or immunoprecipitated (IP)-ULK1 from immunocomplex ULK1 kinase assays from Ctr1 -/- MEFs stably expressing WT or VO treated with VEH or -AA or MEFs stably expressing sgRNA against Rosa (-) or Atp7a ( #1 or #2 ). Quantification: ΔP-ATG13/T-ATG13 normalized to WT, VEH control or Rosa ( - ) control. k , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from Ctr1 -/- MEFs stably expressing WT or VO treated with VEH, -AA, or rapamycin (RAP), with or without bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN normalized to WT, VEH control. l , Scatter dot plot of flow cytometry analysis of autophagic flux quantified by the ratio of mean mCherry-LC3 fluorescent intensity (FI)/mean EGFP-LC3 FI ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and mCherry-EGFP-LC3B treated with VEH, -AA, or RAP, with or without BAF normalized to WT, VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Four asterisks, P<0.0001. n≥7. m , Representative fluorescence images of EGFP, mCherry, or the merge from Ctr1 -/- MEFs stably expressing WT or VO and mCherry-EGFP-LC3B treated with VEH, BAF, - AA, or RAP. n , Scatter dot plot of quantified mCherry positive ( + )-LC3 puncta per cell or mCherry + EGFP + -LC3 puncta per cell ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and mCherry-EGFP-LC3B treated with VEH, BAF, -AA, or RAP. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001; two asterisks, P<0.0001. n≥24. o , Immunoblot detection of LC3-I, LC3-II, P-ERK1/2, T-ERK1/2, or β-ACTIN from MEFs stably expressing sgRNA against Rosa (-) or Atp7a ( #1 or #2 ) treated with VEH or BAF. Quantification: ΔLC3-II/T-β-ACTIN and ΔP-ERK1/2/T-ERK1/2 normalized to Rosa ( - ), VEH control. p , Scatter dot plot of flow cytometry analysis of autophagic flux quantified by the ratio of mean mCherry-LC3 FI/mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-, black circles) or Atp7a ( #1 or #2 , blue squares, blue triangles) and mCherry-EGFP-LC3B treated with VEH or BAF normalized to Rosa ( - ), VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Four asterisks, P<0.0001. n=9. q , Representative fluorescence images of EGFP, mCherry, or the merge from MEFs stably expressing sgRNA against Rosa (-) or Atp7a ( #1 or #2 ) and mCherry-EGFP-LC3B treated with VEH or BAF. r , Scatter dot plot of quantified mCherry + -LC3 puncta per cell or mCherry + EGFP + -LC3 puncta per cell ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-, black circles) or Atp7a ( #1 or #2 , blue squares, blue triangles) and mCherry-EGFP-LC3B treated with VEH or BAF. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Two asterisks, P<0.01; Four asterisks, P<0.0001. n≥26. Western blots images are representative of at least three biological replicates.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Sequencing, Binding Assay, Western Blot, Recombinant, In Vitro, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , Representative live cell imaging of the Cu probe CF4 every ten minutes for 60 minutes from MEFs treated with vehicle (VEH) or amino acid deprivation (-AA). b , Quantification of mean CF4 fluorescence intensity (FI) ± s.e.m. versus time (minutes, min) from MEFs treated with VEH (black circles) or -AA (blue squares) normalized to t=0, five minutes. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Four asterisks, P<0.0001. n=30. c , d , e , f , Representative fluorescence images of EGFP-ATG13 ( c ), EGFP-FIP200 ( d ), EGFP-WIP1 ( e ), or EGFP-DFCP1 ( f ) from Ctr1 -/- MEFs stably expressing CTR1 WT (WT) or empty vector (VO) and EGFP-ATG13 , EGFP-FIP200 , EGFP-WIP1 , or EGFP-DFCP1 treated with VEH or -AA with or without bafilomycin (BAF). g , h , i , j , Scatter dot plot of quantified EGFP + puncta per cell ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and EGFP-ATG13 ( g ), EGFP-FIP200 ( h ), EGFP-WIP1 ( i ), or EGFP-DFCP1 ( j ) treated with VEH or -AA with or without bafilomycin (BAF). Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. EGFP-ATG13, ≥26; EGFP-FIP200, n≥21; EGFP-WIP1, n≥18; EGFP-DFCP1, n≥24. k , Scatter dot plot of flow cytometry analysis of the number of LC3-II positive autophagosomes quantified by the mean EGFP-LC3 FI ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and EGFP-LC3B treated with VEH, -AA, or RAP with or without BAF normalized to WT, VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Four asterisks, P<0.0001. n=12. l , Scatter dot plot of flow cytometry analysis of the number of LC3-II positive autophagosomes quantified by the mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-, black circles) or Atp7a ( #1 or #2 , blue squares, blue triangles) and EGFP-LC3B treated with VEH or BAF normalized to Rosa ( - ), VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001; Four asterisks, P<0.0001. n=9.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Live Cell Imaging, Fluorescence, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , Immunoblot detection of recombinant HA-ULK1 WT or C u b inding m utant HA-ULK1 CBM bound to a resin charged with or without Cu compared to input. b , Immunoblot detection of recombinant phosphorylated (P)-ATG13, total (T)-ATG13, and HA-ULK1 WT or HA-ULK1 CBM from ULK1 in vitro kinase assays. c , Immunoblot detection of recombinant P-ATG13, T-ATG13, or immunoprecipitated (IP)-ULK1 from Ulk1/2 -/- MEFs stably expressing HA-ULK1 WT (WT) or HA-ULK1 CBM (CBM) treated with amino acid deprivation (-AA). d , Immunoblot detection of LC3-I, LC3-II, P-ULK1, T-ULK1, P-ATG13, T-ATG13, or β-ACTIN from MEFs stably expressing sgRNA against Rosa (-) reconstituted with empty vector (VO) or sgRNA against Ulk1 and Ulk2 reconstituted with empty vector (VO), HA-ULK1 WT (WT), or HA-ULK1 CBM (CBM) treated with vehicle (VEH) or -AA with or without bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN and ΔP-ATG13/T-ATG13 normalized to Rosa (-), VO, VEH control. e , Scatter dot plot of flow cytometry analysis of autophagic flux quantified by the ratio of mean mCherry-LC3 fluorescent intensity (FI)/mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangles), or CBM (red inverted triangles) and mCherry-EGFP-LC3B treated with VEH or -AA with or without BAF normalized to Rosa ( - ), VO, VEH control. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05; Three asterisks, P<0.001; Four asterisks, P<0.0001. n=9. f , g , Representative fluorescence images of EGFP-FIP200 ( f ) or EGFP-WIP1 ( g ) from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO, WT, or CBM and EGFP-FIP200 or EGFP-WIP1 treated with VEH or - AA with or without BAF. h , i , Scatter dot plot of quantified EGFP + puncta per cell ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangles), or CBM (red inverted triangles) and EGFP-FIP200 ( h ) or EGFP-WIP1 ( i ) treated with VEH or -AA with or without BAF. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. EGFP-FIP200, n≥26; EGFP-WIP1, n≥27. j , Scatter dot plot of flow cytometry analysis of the number of LC3-II positive autophagosomes quantified by the mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangles), or CBM (red inverted triangles) and EGFP-LC3B treated with VEH or -AA with or without BAF normalized to Rosa ( - ), VO, VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Three asterisks, P<0.001; Four asterisks, P<0.0001. n=9. k , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from Ctr1 flox / flox ( fl/fl ) or Ctr1 -/- ( -/- ) MEFs stably expressing sgRNA against Rosa (-) or sgRNA against Ulk1 and Ulk2 ( + ) treated with VEH or -AA with or without BAF. Quantification: ΔLC3-II/β-ACTIN normalized to Ctr1 flox/flox , Rosa (-), VEH control. Western blot images are representative of at least three biological replicates.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Western Blot, Recombinant, In Vitro, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from Kras G12D ;p53 flox/flox (KP) lung adenocarcinoma cell line #2474 (KP #2474) stably expressing sgRNA against Rosa (-) reconstituted with empty vector (VO) or sgRNA against Ulk1 and Ulk2 reconstituted with empty vector (VO), HA-ULK1 WT (WT), or HA-ULK1 CBM (CBM) treated with vehicle (VEH) or amino acid deprivation (-AA) with or without bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN normalized to Rosa (-), VO, VEH control. b , Immunoblot detection of phosphorylated (P)-ULK1, total (T)-ULK1, P-ATG13, T-ATG13, or β-ACTIN from Kras G12D ;p53 flox/flox (KP) lung adenocarcinoma cell line #2474 cells stably expressing sgRNA against Rosa (-) reconstituted with VO or sgRNA against Ulk1 and Ulk2 reconstituted with VO, WT, or CBM. Quantification: ΔP-ATG13/T-ATG13 normalized to Rosa (-), VO, VEH control. c , Mean tumor volume (mm 3 ) ± s.e.m. versus time (days) in mice injected with KP #2474 cells stably expressing sgRNA against Rosa reconstituted with VO (dark grey line, dark grey circles) , Atg5 (pink line, pink hexagons), or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey line, grey squares), WT (black line, black triangle), or CBM (red line, red inverted triangle). Results were compared using a paired, two-tailed Student’s t-test. One asterisk, P<0.05. n=4. d , Representative dissected tumors from mice injected with KP #2474 cells stably expressing sgRNA against Rosa (-) reconstituted with VO, sgRNA against Atg5 , or sgRNA against Ulk1 and Ulk2 reconstituted with VO, WT, or CBM. e , Scatter dot plot of mean tumor weight (g) ± s.e.m. of tumors at endpoint from KP #2474 cells stably expressing sgRNA against Rosa reconstituted with VO (dark grey circles), Atg5 (pink hexagons), or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangle), or CBM (red inverted triangle). Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. Three asterisks, P<0.001; Four asterisks, P<0.0001. n=4. f , Representative crystal violet images of KP #2474 cells stably expressing sgRNA against Rosa , Ctr1 ( #2 ), Ulk1 and Ulk2 , or Ulk1 , Ulk2 , and Ctr1 (#2 ) from days 3, 4, and 5 of recovery. g , Scatter dot plot of mean absorbance of extracted crystal violet at 590nM ± s.e.m. of KP #2474 cells stably expressing sgRNA against Rosa (black circles) , Ctr1 (#2, pink squares), Ulk1 and Ulk2 (grey triangles), or Ulk1 , Ulk2 , and Ctr1 (#2 , red inverted triangles) from days 3, 4, and 5 of recovery normalized to Rosa , day 3 control. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. n≥9. Western blot images are representative of at least three biological replicates.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Injection, Two Tailed Test