Journal: Genes & Development

Article Title: p62/SQSTM1 synergizes with autophagy for tumor growth in vivo

doi: 10.1101/gad.237354.113

Figure Lengend Snippet: Autophagy disruption by deletion of FIP200 impairs growth of established tumors. ( A ) Schematics for the deletion of floxed FIP200 induced by TAM after transplantation of transformed FIP200 f/f ;CreER MEFs, as described in the Materials and Methods. ( B ) Transformed FIP200 f/f ;CreER MEFs were injected subcutaneously into athymic nude mice, animals ( n = 4 each) were treated with vehicle control (−TAM, black line) or TAM (+TAM, red line) by intraperitoneal (i.p.) injection, and single tumor growth was measured at the indicated time points. Data points represent means of fold change and SD in tumor volume relative to 1 d after the last TAM injection. Representative tumor burden images at the final time point are shown at the right . (***) P < 0.001. ( C ) Tumor lysates harvested from the recipient mice at the final time point were analyzed by Western blotting for various proteins using vinculin as a loading control, as indicated. ( D ) Representative images of tumor sections harvested from the recipient mice at the final time point and analyzed by immunohistochemistry or immunofluorescence with anti-p62, anti-ubiquitin (anti-Ub), anti-BrdU, and anti-caspase-3 (Casp3) antibodies. Bar, 100 μm. ( E , F ) Transformed FIP200 f/f ;CreER MEFs were treated with 4-OHT to activate Cre recombinase to produce FIP200 knockout (KO) cells or with mock medium to generate control (Ctrl) cells, respectively (see Supplemental Fig. S1). Cell viability was determined after 6 h with or without HBSS starvation in E . Cell lysates were analyzed by Western blotting for proteins as indicated after 3 h with or without HBSS starvation in F . ( G ) Immortalized FIP200 f/f ;PyMT;CreER tumor cells were injected into the mammary fat pads of nude mice. Animals ( n = 4 each) were treated with vehicle control (black line) or TAM (red line) by i.p. injection, and single tumor growth was measured at the indicated time points. Data points represent means of fold change and SD in tumor volume relative to 1 d after the last TAM injection. (***) P < 0.001.

Article Snippet: The plasmids TRE·IkBSR-Flag Pgk·Cre encoding IkBSR (Addgene plasmid 22504; Dr. Tyler Jacks), TRMPVIR (Addgene plasmid 27994; Dr. Scott Lowe), HA-p62 (Addgene plasmid 28027; Dr. Qing Zhong), and pEGFP-N1-TFEB (Addgene plasmid 38119; Dr. Andrea Ballabio) were all obtained from Addgene. cDNA fragments for p62 and IkBSR were prepared by PCR using the respective plasmids as templates and then inserted into the BamHI/EcoRI sites of TRMPVIR, which allows inducible expression of these genes upon Dox treatments.

Techniques: Disruption, Transplantation Assay, Transformation Assay, Injection, Control, Western Blot, Immunohistochemistry, Immunofluorescence, Ubiquitin Proteomics, Knock-Out

Journal: Genes & Development

Article Title: p62/SQSTM1 synergizes with autophagy for tumor growth in vivo

doi: 10.1101/gad.237354.113

Figure Lengend Snippet: p62 Knockdown decreases the growth of autophagy-deficient tumors. ( A ) Various transformed MEFs were treated with 250 nM 4-OHT to delete FIP200 and then cultured for 2 d with or without 1 μg/mL Dox, as indicated. Cell lysates were analyzed by Western blotting for the indicated proteins. ( B ) Various transformed MEFs (5 × 10 4 ) were seeded into six-well plates, and the percentage (normalized to cells under −4-OHT/−Dox conditions) of the number of cells was determined after 5 d in DMEM + 10% FBS culture medium under various conditions, as indicated. ( C ) Various transformed MEFs were injected subcutaneously into athymic nude mice, all animals ( n = 4 each) were treated with TAM by i.p. injection for FIP200 deletion and fed without (black line) or with (red line) Dox-containing food, and single tumor growth was measured at the indicated time points. Data points represent means of fold change and SD in tumor volume relative to 1 d after the last TAM injection. (***) P < 0.001. ( D ) Representative images of tumor sections harvested from the recipient mice at the final time point and analyzed by immunohistochemistry with anti-p62 and anti-ubiquitin (anti-Ub) antibodies. Arrows mark p62-positive and ubiquitin-positive aggregates. Bar, 100 μm.

Article Snippet: The plasmids TRE·IkBSR-Flag Pgk·Cre encoding IkBSR (Addgene plasmid 22504; Dr. Tyler Jacks), TRMPVIR (Addgene plasmid 27994; Dr. Scott Lowe), HA-p62 (Addgene plasmid 28027; Dr. Qing Zhong), and pEGFP-N1-TFEB (Addgene plasmid 38119; Dr. Andrea Ballabio) were all obtained from Addgene. cDNA fragments for p62 and IkBSR were prepared by PCR using the respective plasmids as templates and then inserted into the BamHI/EcoRI sites of TRMPVIR, which allows inducible expression of these genes upon Dox treatments.

Techniques: Knockdown, Transformation Assay, Cell Culture, Western Blot, Injection, Immunohistochemistry, Ubiquitin Proteomics

Journal: Genes & Development

Article Title: p62/SQSTM1 synergizes with autophagy for tumor growth in vivo

doi: 10.1101/gad.237354.113

Figure Lengend Snippet: Ectopic p62 expression increases the growth of autophagy-deficient tumor. ( A ) Various transformed MEFs were treated with or without 250 nM 4-OHT and then cultured for 2 d with or without 1 μg/mL Dox, as indicated. Cell lysates were analyzed by Western blotting for the indicated proteins. ( B ) ip62 cells (5 × 10 4 ) were seeded into six-well plates, and the number of cells was determined after 3 or 6 d in DMEM + 10% FBS culture medium under various conditions, as indicated. ( C ) ip62 cells were injected subcutaneously into athymic nude mice, animals ( n = 4 each) were treated with or without TAM by i.p. injection for FIP200 deletion and fed without (black line) or with (red line) Dox-containing food, and single tumor growth was measured at the indicated time points. Data points represent means of fold change and SD in tumor volume relative to 1 d after the last TAM injection. (***) P < 0.001. ( D ) Representative images of tumor sections harvested from the recipient mice at the final time point and analyzed by immunohistochemistry with anti-p62 and anti-ubiquitin (anti-Ub) antibodies. Arrows mark p62-positive and ubiquitin-positive aggregates. Bar, 100 μm. ( E ) Lysates were prepared from mammary tumors of recipient mice and then analyzed by Western blotting for the indicated proteins. ( F ) The proliferation and apoptosis of tumor cells in recipient mice were analyzed by BrdU incorporation assays as described in Materials and Methods ( top panels) or immunohistochemistry using anti-cleaved caspase-3 ( bottom panels), respectively. Bar, 100 μm. Data at the right represent mean ± SD ( n = 4 for each sample) of relative numbers of BrdU-positive or cleaved caspase-3-positive cells per field.

Article Snippet: The plasmids TRE·IkBSR-Flag Pgk·Cre encoding IkBSR (Addgene plasmid 22504; Dr. Tyler Jacks), TRMPVIR (Addgene plasmid 27994; Dr. Scott Lowe), HA-p62 (Addgene plasmid 28027; Dr. Qing Zhong), and pEGFP-N1-TFEB (Addgene plasmid 38119; Dr. Andrea Ballabio) were all obtained from Addgene. cDNA fragments for p62 and IkBSR were prepared by PCR using the respective plasmids as templates and then inserted into the BamHI/EcoRI sites of TRMPVIR, which allows inducible expression of these genes upon Dox treatments.

Techniques: Expressing, Transformation Assay, Cell Culture, Western Blot, Injection, Immunohistochemistry, Ubiquitin Proteomics, BrdU Incorporation Assay

Journal: Genes & Development

Article Title: p62/SQSTM1 synergizes with autophagy for tumor growth in vivo

doi: 10.1101/gad.237354.113

Figure Lengend Snippet: p62 regulates NF-kB activation, and inhibition of NF-kB impairs the tumor growth under autophagy-deficient condition. ( A ) Various transformed MEFs were treated with 250 nM 4-OHT to delete FIP200 and then cultured for 2 d with or without 1 μg/mL Dox to induce p62 knockdown ( left four lanes) or ectopic p62 expression ( right two lanes), as indicated. Cell lysates were analyzed by Western blotting for the indicated proteins. ( B ) Various transformed MEFs were treated with 250 nM 4-OHT to delete FIP200 and then cultured for 2 d with or without 1 μg/mL Dox, as indicated. Cytoplasmic (Cyto) and nuclear (Nucl) fractions were then prepared and analyzed by Western blotting for the indicated proteins. ( C ) ip62 cells were treated with 250 nM 4-OHT for deletion of FIP200 and then cultured for 2 d with or without 1 μg/mL Dox. RNA was isolated from cells and subjected to the analysis by quantitative RT–PCR (qRT–PCR) to detect the expression of NF-kB target genes, as indicated. The mean ± SD of relative levels (normalized to no Dox treatment) is shown. ( D ) Various transformed MEFs were treated with 4-OHT to delete FIP200 and then cultured overnight with or without 1 μg/mL Dox, as indicated. Cell lysates were analyzed by Western blotting for the indicated proteins. ( E ) iIkBSR cells were treated with 250 nM 4-OHT to delete FIP200 and then cultured for 2 d with or without 1 μg/mL Dox, as indicated. Cytoplasmic (Cyto) and nuclear (Nucl) fractions were prepared and analyzed by Western blotting for the indicated proteins. ( F ) iIKBSR cells were treated with 250 nM 4-OHT for deletion of FIP200 and then cultured for 2 d with or without 1 μg/mL Dox. RNA was isolated from cells and subjected to the analysis by qRT–PCR to detect the expression of NF-kB target genes, as indicated. The mean ± SD of relative levels (normalized to no Dox treatment) is shown. ( G ) iIkBSR cells (5 × 10 4 ) were seeded into six-well plates, and the percentage (normalized to cells under −4-OHT/−Dox conditions) of the number of cells was determined after 5 d in DMEM + 10% FBS culture medium under various conditions, as indicated. ( H ) iIkBSR cells were injected subcutaneously into athymic nude mice, animals ( n = 4 each) were treated with or without TAM by i.p. injection for FIP200 deletion and fed without (black line) or with (red line) Dox-containing food, and single tumor growth was measured at the indicated time points. Data points represent means of fold change and SD in tumor volume relative to 1 d after the last TAM injection. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Article Snippet: The plasmids TRE·IkBSR-Flag Pgk·Cre encoding IkBSR (Addgene plasmid 22504; Dr. Tyler Jacks), TRMPVIR (Addgene plasmid 27994; Dr. Scott Lowe), HA-p62 (Addgene plasmid 28027; Dr. Qing Zhong), and pEGFP-N1-TFEB (Addgene plasmid 38119; Dr. Andrea Ballabio) were all obtained from Addgene. cDNA fragments for p62 and IkBSR were prepared by PCR using the respective plasmids as templates and then inserted into the BamHI/EcoRI sites of TRMPVIR, which allows inducible expression of these genes upon Dox treatments.

Techniques: Activation Assay, Inhibition, Transformation Assay, Cell Culture, Knockdown, Expressing, Western Blot, Isolation, Quantitative RT-PCR, Injection