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Shanghai GenePharma ctr1 plasmids
Ctr1 Plasmids, supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

ctr1 plasmids - by Bioz Stars, 2026-05

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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.

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

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.

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.

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

a , Normalized representative images of in vivo luminescence of Kras G12D/+ ; Tr p 53 flox/flox ;Rosa26::LSL-Luc ( KPLuc ) mice introduced with either sgRNA against β-GAL or Ctr1 at indicated time points. b , c , d , e , f , g , h , i , Representative images of H&E stained ( b -5x stitched images, d -5x); immunohistochemical detection of CCS ( c -5x stitched images, e -5x), LC3 ( f -40x), p62 ( g -40x), or Ki67 ( i -40x); or electron microscopy (EM) ( h ) of lungs from KPLuc mice expressing sgRNA against β-GAL or Ctr1 (5x scale bar: 250 μm; 40x scale bar: 50 μm). j , k , l , m , n , o , Scatter dot plot of mean ± s.e.m. % abnormal lung tissue ( j , β-GAL , n=36 and Ctr1 , n=35), % CCS positive staining per tumor ( k , β-GAL , n=35 and Ctr1 , n=36), % LC3 positive staining per tumor ( l , n=35), % p62 positive staining per tumor ( m , n=35), autophagosome number per tumor cell ( n , β-GAL , n=31 and Ctr1 , n=30), or % Ki67 positive staining per tumor ( o , n=36) from KPLuc mice expressing sgRNA against β-GAL (black circles) or Ctr1 (red squares ) . Results were compared using an unpaired, one-tailed Student’s t-test. Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. p , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from KP lung adenocarcinoma cell lines #54 (KP #54) and #2474 (KP #2474) stably expressing sgRNA against Rosa (-) or Ctr1 (#1 or #2 ) treated with vehicle (VEH) or bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN normalized to Rosa ( - ), VEH control. q , Schematic of clonogenic survival assay in response to starvation. KP #54 and KP #2474 cells stably expressing sgRNA against Rosa, Atg5, or Ctr1 following one day of starvation (EBSS) were recovered for three days in normal medium (DMEM). r , Representative crystal violet images of KP #54 and KP #2474 cells stably expressing sgRNA against Rosa, Atg5, or Ctr1 ( #1 or #2 ) from days 3, 4, and 5 of recovery. s , Scatter dot plot of mean absorbance of extracted crystal violet at 590nM ± s.e.m. of KP #54 and KP #2474 cells stably expressing sgRNA against Rosa (black circles) , Atg5 (grey inverted triangles), or Ctr1 ( #1 or #2, pink squares, red 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. One asterisk, P<0.05; Two asterisks, P<0.01; Four asterisks, P<0.0001. KP#54, n≥11; KP #2474, n=12. Western blot images are representative of at least three biological replicates.

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 , Normalized representative images of in vivo luminescence of Kras G12D/+ ; Tr p 53 flox/flox ;Rosa26::LSL-Luc ( KPLuc ) mice introduced with either sgRNA against β-GAL or Ctr1 at indicated time points. b , c , d , e , f , g , h , i , Representative images of H&E stained ( b -5x stitched images, d -5x); immunohistochemical detection of CCS ( c -5x stitched images, e -5x), LC3 ( f -40x), p62 ( g -40x), or Ki67 ( i -40x); or electron microscopy (EM) ( h ) of lungs from KPLuc mice expressing sgRNA against β-GAL or Ctr1 (5x scale bar: 250 μm; 40x scale bar: 50 μm). j , k , l , m , n , o , Scatter dot plot of mean ± s.e.m. % abnormal lung tissue ( j , β-GAL , n=36 and Ctr1 , n=35), % CCS positive staining per tumor ( k , β-GAL , n=35 and Ctr1 , n=36), % LC3 positive staining per tumor ( l , n=35), % p62 positive staining per tumor ( m , n=35), autophagosome number per tumor cell ( n , β-GAL , n=31 and Ctr1 , n=30), or % Ki67 positive staining per tumor ( o , n=36) from KPLuc mice expressing sgRNA against β-GAL (black circles) or Ctr1 (red squares ) . Results were compared using an unpaired, one-tailed Student’s t-test. Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. p , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from KP lung adenocarcinoma cell lines #54 (KP #54) and #2474 (KP #2474) stably expressing sgRNA against Rosa (-) or Ctr1 (#1 or #2 ) treated with vehicle (VEH) or bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN normalized to Rosa ( - ), VEH control. q , Schematic of clonogenic survival assay in response to starvation. KP #54 and KP #2474 cells stably expressing sgRNA against Rosa, Atg5, or Ctr1 following one day of starvation (EBSS) were recovered for three days in normal medium (DMEM). r , Representative crystal violet images of KP #54 and KP #2474 cells stably expressing sgRNA against Rosa, Atg5, or Ctr1 ( #1 or #2 ) from days 3, 4, and 5 of recovery. s , Scatter dot plot of mean absorbance of extracted crystal violet at 590nM ± s.e.m. of KP #54 and KP #2474 cells stably expressing sgRNA against Rosa (black circles) , Atg5 (grey inverted triangles), or Ctr1 ( #1 or #2, pink squares, red 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. One asterisk, P<0.05; Two asterisks, P<0.01; Four asterisks, P<0.0001. KP#54, n≥11; KP #2474, n=12. Western blot images are representative of at least three biological replicates.

Techniques: In Vivo, Staining, Immunohistochemical staining, Electron Microscopy, Expressing, One-tailed Test, Western Blot, Stable Transfection, Clonogenic Cell Survival Assay

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.

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.

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

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.

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.

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

Fig. 3. ES supplementation rescues the steady-state levels of copper-containing subunits of CcO in mammalian cell lines with genetic defects in copper metabolism. (A) Immunoblot analysis of CTR1 in Ctr1+/+ and Ctr1−/−H9c2 cells. The arrowheads labeled “g” and “t” indicate the full-length glycosylated and truncated forms of CTR1, respectively. (B) Total copper levels measured by ICP-MS in H9c2 cells. Data are presented as mean ± SD (n = 4, two-tailed unpaired Student’s t test, **P < 0.001). (C) Immunoblot analysis of CCS, COX4, and GAPDH protein levels in Ctr1+/+ and Ctr1−/−H9c2 cells. GAPDH serves as a loading control. (D) The Ctr1+/+ and Ctr1−/−H9c2 rat cardiomyocytes and (E) MEFs were cultured for 3 d with the indicated doses of ES followed by Western analysis of COX1 protein levels. ATP5A is used as loading control. (F) Control (MCH46) and SCO2 patient cell lines were cultured for 3 or 6 d in the presence of the indicated concentrations of ES or a copper–histidinate complex (Cu-His) in DMEM with 10% FBS. The cellular COX2 levels were detected by SDS/PAGE/Western blot analysis. β-Actin (ACTB) was used as a loading control.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elesclomol restores mitochondrial function in genetic models of copper deficiency.

doi: 10.1073/pnas.1806296115

Figure Lengend Snippet: Fig. 3. ES supplementation rescues the steady-state levels of copper-containing subunits of CcO in mammalian cell lines with genetic defects in copper metabolism. (A) Immunoblot analysis of CTR1 in Ctr1+/+ and Ctr1−/−H9c2 cells. The arrowheads labeled “g” and “t” indicate the full-length glycosylated and truncated forms of CTR1, respectively. (B) Total copper levels measured by ICP-MS in H9c2 cells. Data are presented as mean ± SD (n = 4, two-tailed unpaired Student’s t test, **P < 0.001). (C) Immunoblot analysis of CCS, COX4, and GAPDH protein levels in Ctr1+/+ and Ctr1−/−H9c2 cells. GAPDH serves as a loading control. (D) The Ctr1+/+ and Ctr1−/−H9c2 rat cardiomyocytes and (E) MEFs were cultured for 3 d with the indicated doses of ES followed by Western analysis of COX1 protein levels. ATP5A is used as loading control. (F) Control (MCH46) and SCO2 patient cell lines were cultured for 3 or 6 d in the presence of the indicated concentrations of ES or a copper–histidinate complex (Cu-His) in DMEM with 10% FBS. The cellular COX2 levels were detected by SDS/PAGE/Western blot analysis. β-Actin (ACTB) was used as a loading control.

Article Snippet: A CRISPR/Cas9-mediated Ctr1-KO rat H9c2 cell line was generated by using lentiCRISPR v2 plasmid (no. 52961; Addgene).

Techniques: Western Blot, Labeling, Two Tailed Test, Control, Cell Culture, SDS Page

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