cb105 atg5 shrna mouse lentiviral particles santa cruz  (Santa Cruz Biotechnology)

Journal: Autophagy

Article Title: Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance

doi: 10.1080/15548627.2023.2300913

Figure Lengend Snippet: Mutual regulation of CAF autophagy and activation in pancreatic cancer. (A) multi-IHC staining for MAP1LC3B, ATG5, SQSTM1, ACTA2, FAP and DAPI in PDAC. (B and C) tissue microarray analysis of the prognostic role of MAP1LC3B staining intensity in cancer cells and CAFs in pancreatic cancer. (D and E) MAP1LC3B, SQSTM1, CD8A IHC and Sirius red staining in human PDAC tissues and quantification of collagen deposition using Sirius red staining and the CD8A-positive cell area per field. (F and G) Representative transmission electron microscopy images and statistical results of autophagosomes and autolysosomes in CAFs after adding ATRA (1 mM) or PBS. (H) Western blot analysis of LC3-I, LC3-II, SQSTM1, FAP and ACTA2 in CAFs and PSC (CAFs treated after ATRA) with or without CQ. (I and J) Representative microphotographs and statistical results of MAP1LC3B immunofluorescence staining in CAFs following CQ treatment. The addition of CQ to CAFs induced an accumulation of MAP1LC3B in the cytoplasm. (K) CAFs were subjected to Atg5 KD, followed by IB for the different indicated proteins. (L and M) CAFs were subjected to Atg5 KD, followed by IB for ACTA2 (green) and DAPI (blue). (N) CAFs were treated with CQ and then subjected to IB for the different indicated proteins.

Article Snippet: Atg5 shRNA lentiviral particles(m) , Santa Cruz Biotechnology , sc -41,446-V.

Techniques: Activation Assay, Immunohistochemistry, Microarray, Staining, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence

Journal: Autophagy

Article Title: Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance

doi: 10.1080/15548627.2023.2300913

Figure Lengend Snippet: Genetic inhibition of CAF autophagy induced CD274-upregualtion mediated immune escape in both immune-competent mice and pancreatic cancer cells. (A) schematic protocols of WT-mCafs and Atg5 KD-mCafs with KPC separately and subcutaneously. injected into immunocompetent and immunodeficient mice ( n = 5). (B-G) Representative images displaying tumors, tumor weight, and survival of immunocompetent and immunodeficient mice bearing WT-mCafs and Atg5 KD-mCafs with KPC. (H and I) Representative images and statistical results of tumor-infiltrating lymphocytes and immunomodulators in tumor cells ( n = 5). (J and K) immunoblot analysis of CD274 expression in pancreatic tumors with Atg5 KD-mCafs. (L and M) Representative images and further quantification of tumor-bearing Atg5 KD-CAFs followed by immunofluorescence staining for ACTA2 (green), ATG5 (red), CD274 (pink) and DAPI (blue). (Q and R) Representative images and further quantification of tumor cells cocultured with Atg5 KD-CAFs followed by immunofluorescence staining for CD274 (red) and DAPI (blue). (N) immunoblot analysis of CD274 and MHC-1 expression in tumor cell lines cocultured with WT-CAFs and Atg5 KD-CAFs. (O and P) flow cytometry and further quantification of CD274 expression in tumor cell lines cocultured with WT-CAFs and Atg5 KD-CAFs. (S and T) Representative images and statistical results of T cell-mediated cancer cell-killing assay. KPC cells were pre-cocultured with WT-CAFs and Atg5 KD-CAFs for 24 h, then cocultured with activated T cells for 48 h and subjected to crystal violet staining. The ratio of tumor cells to T cells was 1:8.

Article Snippet: Atg5 shRNA lentiviral particles(m) , Santa Cruz Biotechnology , sc -41,446-V.

Techniques: Inhibition, Injection, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Autophagy

Article Title: Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance

doi: 10.1080/15548627.2023.2300913

Figure Lengend Snippet: Inhibition of CAF autophagy improved the in vivo anti-tumor effect of immunotherapy. (A) schematic protocol for the genetic inhibition of CAF autophagy combined with anti-PDCD1/CD279 antibody. (B-D) Representative images displaying tumors, tumor weight, and mouse weight of atg5 f/f - Acta2creER T2 genetic mice treated with anti-PDCD1/CD279 antibody. (E and F) Representative photograph and statistical results of the IVIS imaging system in mice orthotopically implanted with luciferase-expressing KPC in atg5 f/f - Acta2creER T2 genetic mice. (G-I) flow cytometric analysis and statistical results of lymphocytes that have infiltrated the tumors and membrane CD274 and MHC-1 expression on tumor cells ( n = 7). (J) CyTOF analysis of membrane CD274 and MHC-1 expression on tumor cells in the four treatment groups ( n = 3). (K) the statistical results of tumor weight following pretreatment with anti-CD8A and anti-KLRB1C/NK1.1 in atg5 f/f - Acta2creER T2 genetic mice combined with anti-PDCD1/CD279 antibodies. (L) survival curve of orthotopic tumor implantation in atg5 f/f - Acta2creER T2 genetic mice. atg5 f/f - Acta2creER T2 genetic mice-orthotopic mice were treated with anti-PDCD1/CD279 antibodies until the mice were at the point of death and met the prespecified early removal criteria approved by the IACUC.

Article Snippet: Atg5 shRNA lentiviral particles(m) , Santa Cruz Biotechnology , sc -41,446-V.

Techniques: Inhibition, In Vivo, Imaging, Luciferase, Expressing, Membrane, Tumor Implantation

Journal: Autophagy

Article Title: Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance

doi: 10.1080/15548627.2023.2300913

Figure Lengend Snippet: Deletion of CAF autophagy decreased IL6 secretion, which further increased CD274 expression by the ubiquitin proteasome system in pancreatic tumor cells. (A and B) differential cytokine expression was detected between WT and Atg5 KD CAFs using a cytokine antibody array and further quantified with a heatmap analysis. (C) the concentration of the top five secreted cytokines in the cytokine antibody array was identified by an ELISA. (D and E) flow cytometric analysis and statistical results of CD274 expression in tumor cells with or without tocilizumab under CAF-CM. (F) immunoblot analysis of CD274 expression in tumor cells with or without tocilizumab-treated CAFs. (G and H) Representative images and further quantification of tumor cells with or without tocilizumab-treated CAFs. (I and J) Representative images and statistical results of the T cell-mediated cancer cell-killing assay. KPC cells with or without anti-IL6R pre-cocultured CAFs for 24 h were cocultured with activated T cells for 48 h and subjected to crystal violet staining. The ratio of tumor cells to T cells was 1:8. (K) qRT-PCR examination of CD274 expression in tumor cells cocultured with WT-CAFs and Atg5 KD-CAFs for 24 h. (L) qRT-PCR examination of CD274 expression in tumor cells with or without tocilizumab-treated CAFs. (M) immunoblot analysis of CAFs treated with tocilizumab or anti-IL6R for 24 h with and without chloroquine (40 μM for last 6 h of treatment) or MG132 (20 μM for last 6 h of treatment). (N and O) stability analysis of CD274 in tumor cells treated with or without tocilizumab or anti-IL6R cocultured with CAFs following treatment with CHX. (P) ubiquitination assay of CD274 in tumor cells treated with or without tocilizumab or anti-IL6R and cocultured with CAFs after treatment with MG132.

Article Snippet: Atg5 shRNA lentiviral particles(m) , Santa Cruz Biotechnology , sc -41,446-V.

Techniques: Expressing, Ubiquitin Proteomics, Ab Array, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Quantitative RT-PCR

Journal: Autophagy

Article Title: Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance

doi: 10.1080/15548627.2023.2300913

Figure Lengend Snippet: Transcriptional activation of USP14 by STAT3 interacted with and negatively regulated CD274 in pancreatic cancer. (A and B) heatmap and volcano plot of RNAseq in BxPC-3 with or without tocilizumab treatment cocultured with CAFs for 24 h. (C) intersection analysis of the proteasome-mediated degradation pathway in RNAseq and CD274 flag-IP-LCMS. (D) immunoblot analysis of USP14 expression in tumor cells with or without tocilizumab or anti-IL6R treatment and cocultured with CAFs. (E) immunoblot analysis of USP14 expression in tumor cells cocultured with WT-CAFs and Atg5 KD-CAFs for 48 h. (F) chromatin immunoprecipitation (ChIP) assay analysis of the STAT3-bound potential binding site in the USP14 promotor. (G and H) schematic representation of the USP14 promoter cloned into the pGL4 vector. The predicted STAT3-binding motifs were shown and the promoter constructs containing mutations in this region to cause a STAT3-binding deficiency were generated. (I) cell lysates from SW1990, BxPC-3, and Panc02 were separately analyzed by IP and western blotting using the antibodies indicated. Representative images are shown. (J) GST affinity-isolation assay of USP14-his and GST-CD274 proteins. Representative images are shown. (K and L) Representative images and statistical results of individual immunofluorescence staining of the USP14 and CD274 interaction in KPC cells by a duolink assay. The red dots (USP14/CD274 interaction) indicate their interaction. (M-T) immunoblot analysis, flow cytometric analysis, and immunofluorescence staining of CD274 expression in tumor cells following treatment with IU1 (USP14 inhibitor) and Usp14 knockdown.

Article Snippet: Atg5 shRNA lentiviral particles(m) , Santa Cruz Biotechnology , sc -41,446-V.

Techniques: Activation Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Clone Assay, Plasmid Preparation, Construct, Generated, Isolation, Immunofluorescence, Staining, Knockdown

Journal: Autophagy

Article Title: Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance

doi: 10.1080/15548627.2023.2300913

Figure Lengend Snippet: Key resources table.

Article Snippet: Atg5 shRNA lentiviral particles(m) , Santa Cruz Biotechnology , sc -41,446-V.

Techniques: Ubiquitin Proteomics, Recombinant, Virus, shRNA, Control, Activation Assay, Transfection, Plasmid Preparation, Protease Inhibitor, Staining, Cell Isolation, Luciferase, Reporter Gene Assay, In Situ, Cell Fractionation, Software

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: ATG5 shows opposite effects on cell growth under normal culture conditions and serum starvation conditions (A) ATG5 was depleted or overexpressed in HeLa cells with two ATG5-specific siRNAs (left) or a Flag-tagged ATG5 construct (right), respectively. HeLa cells transfected with a control siRNA (left) or Flag control construct (right) were used as the corresponding controls. Cells were further cultured under normal culture conditions for the indicated durations, and the relative cell growth ability was examined by a cell counting kit 8 (CCK-8) assay. (B) HeLa cells transfected as described in A were cultured under serum starvation conditions for the indicated durations. The relative cell growth ability was examined as described in (A). (C) HeLa cells were transfected with a Flag control or a Flag-tagged ATG5 construct and cultured under normal conditions (left) or serum starvation conditions (right) for 6 h. Cells were then treated with or without 1 μM Baf A1 as indicated and were further cultured for different durations as indicated. The relative cell growth ability was examined by CCK-8 assays as described in (A). (D) HeLa cells stably transfected with an ATG5-specific shRNA or a Flag-tagged ATG5 construct were generated, and a colony formation assay was performed with cells cultured under normal conditions (left upper) or serum starvation conditions (left lower) for 7 days. The colonies were photographed (left), and the relative quantification of the number of colonies is shown (middle and right). (E) HeLa cells were transfected with a Flag control or a Flag-tagged ATG5K130R construct and cultured under normal conditions (left) or serum starvation conditions (right) for 6 h. Cells were then treated with or without 1 μM Baf A1 as indicated and were further cultured for different durations as indicated. The relative cell growth ability was examined by CCK-8 assays as described in (A). Statistics: all experiments were repeated more than 3 times. The bars and error bars indicate the mean ± s.d. values; n = 3 independent replicates. Two-tailed unpaired Student's t test was performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns: nonsignificant.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Construct, Transfection, Control, Cell Culture, Cell Counting, CCK-8 Assay, Stable Transfection, shRNA, Generated, Colony Assay, Quantitative Proteomics, Two Tailed Test

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: ATG5 negatively regulates the protein level of c-Myc, especially under normal culture conditions (A) HeLa cells were transfected with an ATG5-specific siRNA or a Flag-tagged ATG5 construct and were further cultured under normal conditions or serum starvation conditions for 48 h. Western blot analyses or RT-PCR analyses were then performed. (B) 293T cells were transfected with an ATG5-specific siRNA or a Flag-tagged ATG5 construct and were further cultured under normal conditions or serum starvation conditions for 48 h. Western blot analyses were then performed. (C) Flag-tagged ATG5 or ATG5-specific siRNA-transfected 293T cells were further transfected with a control luciferase reporter construct or a c-Myc-targeted luciferase reporter construct as indicated. Cells were then cultured under normal conditions or serum starvation conditions for 48 h. Luciferase activity was evaluated. (D) 293T cells were transfected with an ATG5-specific siRNA or a Flag-tagged ATG5 construct and were further cultured under normal conditions or serum starvation conditions for 48 h as indicated. Cells were then lysed and subjected to quantitative RT-PCR analyses. (E) HeLa cells transfected with a Flag control construct, a Flag-tagged ATG5 construct, or a Flag-tagged ATG5 construct together with an HA-tagged c-Myc construct were cultured under normal conditions for the indicated durations. The relative cell growth ability was examined by CCK-8 assays. (F) HeLa cells were transfected with 3 individual c-Myc-specific siRNAs for 48 h. Western blot analyses were performed to determine the knockdown efficiency of the c-Myc siRNAs (left). HeLa cells transfected with a control siRNA, an ATG5-specific siRNA, or an ATG5-specific siRNA together with a c-Myc-specific siRNA as indicated were cultured under normal conditions for the indicated durations. The relative cell growth ability was examined by CCK-8 assays (right). (G) Predicted model. Under normal culture conditions, ATG5 negatively regulates c-Myc and further affects the expression of c-Myc downstream target genes and cell growth. Statistics: all experiments were repeated more than 3 times. The bars and error bars indicate the mean ± s.d. values; n = 3 independent replicates. Two-tailed unpaired Student's t test was performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Transfection, Construct, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Luciferase, Activity Assay, Quantitative RT-PCR, CCK-8 Assay, Knockdown, Expressing, Two Tailed Test

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: ATG5 regulates c-Myc protein degradation through the ubiquitination-proteasome pathway (A) 293T cells were transfected with a Flag control or a Flag-tagged ATG5 construct for 48 h. Cells were then treated with or without 1 μM Baf A1 (bafilomycin A1), 25 μM MG132, or both as indicated for another 10 h. Cell extracts were prepared, and western blot analyses were performed. (B) HeLa and 293T cells transfected with a Flag control construct or a Flag-tagged ATG5 construct were treated with 50 μg/mL CHX (cycloheximide) for the indicated durations (chase assays). Cells were then lysed and subjected to western blot analyses. Relative protein levels of c-Myc were analyzed with ImageJ software (right). (C) 293T cells transfected with a control siRNA or an ATG5-specific siRNA were treated with 50 μg/mL CHX for the indicated durations (chase assays). Cells were then lysed and subjected to western blot analyses. Relative protein levels of c-Myc were analyzed with ImageJ software (right). (D) An HA-tagged c-Myc and an His-tagged ubiquitin construct were transfected with or without a Flag-tagged ATG5 construct into 293T cells as indicated for 48 h. Cells were then treated with or without 25 μM MG132 for another 10 h. Cells were lysed under denaturing conditions, and the c-Myc protein was immunoprecipitated (IP) with c-Myc-specific antibodies. IP with immunoglobulin G (IgG) antibodies was performed as a negative control. The precipitated proteins were subjected to western blot analyses with the indicated antibodies. Statistics: all experiments were repeated more than 3 times. The bars and error bars indicate the mean ± s.d. values; n = 3 independent replicates.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Ubiquitin Proteomics, Transfection, Control, Construct, Western Blot, Software, Immunoprecipitation, Negative Control

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: ATG5 interacts with c-Myc under normal culture conditions (A) 293T cells were transfected with a Flag-tagged ATG5 and an HA-tagged c-Myc constructs for 48 h. Cells were lysed and subjected to coimmunoprecipitation (co-IP) assays with antibodies against Flag (left) or HA (right) as indicated. (B) Co-IP assays were performed in 293T cells with antibodies against ATG5 (left) or c-Myc (right) to determine the interaction between endogenous ATG5 and c-Myc proteins. (C) 293T cells were transfected with a GFP-tagged ATG5 and an HA-tagged c-Myc construct for 48 h under normal or serum starvation conditions. Immunofluorescence (IF) analysis was then performed to evaluate the localization of ATG5 (green) and c-Myc (red). The blue signal indicates the nucleolus (stained with DAPI). (D) GST-tagged ATG5 and His-tagged c-Myc were expressed in E coli and purified (left two panels). The two proteins were co-incubated, and IP assays with antibodies against ATG5 or c-Myc (right two panels) were performed as indicated to evaluate the direct binding between ATG5 and c-Myc in vitro . (E) 293T cells co-transfected with a GFP-tagged ATG5 and an HA-tagged c-Myc were further cultured under normal conditions or serum starvation conditions as indicated. Cell extracts were prepared, and co-IP assays with antibodies against GFP were performed. Co-IP with IgG antibodies was performed as a negative control. (F) 293T cells transfected with a flag-tagged ATG5 and a GFP-tagged LC3 were cultured under normal conditions or serum starvation conditions as indicated. IF analysis was performed to evaluate the localization of ATG5 (red) and LC3 (green). DAPI staining was used to indicate the nucleolus (blue). (G) Molecular model. ATG5 interacts with c-Myc and promotes c-Myc protein degradation under normal culture conditions. However, when cells are subjected to serum starvation to activate autophagy, ATG5 may be released from c-Myc and mainly contribute to autophagy.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Transfection, Construct, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Purification, Incubation, Binding Assay, In Vitro, Cell Culture, Negative Control

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: ATG5 negatively regulates c-Myc independent of its conjugation with ATG12 (A) 293T cells were transfected with a Flag-tagged ATG5K130R and an HA-tagged c-Myc construct for 48 h. Cells were lysed and subjected to co-immunoprecipitation (co-IP) assays with antibodies against Flag (left) or HA (right) as indicated. (B) 293T cells transfected with a Flag control and a Flag-tagged ATG5K130R construct were further cultured under normal conditions or serum starvation conditions for 48 h. Western blot analyses were then performed. (C) 293T cells were transfected with a Flag-tagged ATG5 construct or an ATG12-specific siRNA or together for 48 h. Western blot analyses were then performed. (D) An HA-tagged c-Myc and an His-tagged ubiquitin construct were transfected with a Flag-tagged ATG5 or ATG5K130R mutant construct into 293T cells as indicated for 48 h. Cells were lysed under denaturing conditions, and the c-Myc protein was immunoprecipitated (IP) with HA-tag-specific antibodies. IP with IgG antibodies was performed as a negative control. The precipitated proteins were subjected to western blot analyses with the indicated antibodies.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Conjugation Assay, Transfection, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Cell Culture, Western Blot, Ubiquitin Proteomics, Mutagenesis, Negative Control

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: ATG5 recruits the E3 protein-ubiquitin ligase FBW7 to bind c-Myc (A) 293T cells were transfected with a Flag-tagged ATG5 for 48 h. Cells were lysed, and co-IP assays were performed with antibodies against Flag. IgG antibodies were used as a negative control. (B) 293T cells were transfected with an HA-tagged c-Myc construct together with increasing amounts of a Flag-tagged ATG5 construct for 48 h. Cells were lysed, and co-IP assays were performed with antibodies against HA. IgG antibodies were used as a negative control. (C) 293T cells were transfected with an ATG5-specific siRNA or a control siRNA for 48 h. Cells were lysed, and co-IP assays were performed with antibodies against c-Myc. IgG antibodies were used as a negative control. (D) GST-tagged ATG5 and His-tagged FBW7 were expressed in E coli and purified (upper). The two proteins were co-incubated, and IP assays with antibodies against ATG5 or His (lower) were performed as indicated to evaluate the direct binding between ATG5 and FBW7 in vitro . (E) A FBW7-specific siRNA was transfected into 293T cells for 48 h. Cells were then lysed, and the RNAi efficiency was determined by western blot analysis (upper). 293T cells were transfected with a Flag-tagged ATG5 construct, a HA-tagged FBW7 construct, FBW7-specific siRNA, or distinct combinations as indicated for 48 h. Cell extracts were prepared, and western blot analyses were performed (lower). (F) 293T cells were cultured under normal conditions or serum starvation conditions as indicated. Cells were harvested, and co-IP assays were performed with antibodies against c-Myc. IgG antibodies were used as a negative control. (G) Molecular model. Under normal culture conditions, ATG5 recruits FBW7 to c-Myc and further promotes c-Myc protein degradation. However, under serum starvation conditions, ATG5 may mainly contribute to the regulation of autophagy.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay, Negative Control, Construct, Control, Purification, Incubation, Binding Assay, In Vitro, Western Blot, Cell Culture

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: The observed ATG5-c-Myc regulatory axis is involved in the regulation of mouse embryonic stem cell (ESC) differentiation (A) An Oct4-EGFP mouse ES (Oct4-EGFP-mES) cell line was used to establish the ESC differentiation system. Briefly, ESCs were cultured for the indicated durations in differentiation medium from which LIF (leukemia inhibitory factor) was removed and to which RA (retinoic acid, 1 μM) was added. The differentiation efficiency was determined by analyzing the morphologies of the colonies (upper left), the expression of Oct4-regulated EGFP (upper right), and the staining of AP (alkaline phosphatase) (lower). (B) Oct4-EGFP-mESCs were differentiated for 4 days as described in (A). IF analyses (left) were then performed to determine the levels of LC3 (red) and GFP-Oct4 (green). DAPI staining indicates the nucleolus (blue). Western blot analyses were also performed with antibodies as indicated (right). (C) Oct4-EGFP-mESCs were differentiated as described in (A). Western blot and RT-PCR analyses were performed with antibodies as indicated. (D) Oct4-EGFP-mESCs were differentiated for 4 days as described in (A). Cells were then treated with 25 μM MG132 for 6 h. Western blot analyses were performed with antibodies as indicated. (E) Oct4-EGFP-mESCs were stably transfected with an ATG5-specific shRNA or a control shRNA. Cells were then differentiated as described in A by removing LIF and adding RA. Western blot analyses were performed with antibodies as indicated. (F) Oct4-EGFP-mESCs were differentiated as described in (A). Cells were then treated with 25 μM MG132 for 6 h. Co-IP assays with antibodies against ATG5 were performed. IgG antibodies were used as a negative control. (G) Oct4-EGFP-mESCs were stably transfected with an ATG5-specific shRNA, an ATG5-specific shRNA combined with a c-Myc-specific shRNA, or a control shRNA as indicated. Cells were then differentiated for 4 days as described in A by removing LIF and adding RA. AP staining assays were performed to determine the differentiation efficiency.

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Cell Culture, Expressing, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, shRNA, Control, Co-Immunoprecipitation Assay, Negative Control

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: ATG5 shRNA (mouse) Lentiviral Particles , Santa Cruz , sc-41446-V.

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription