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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 1. AKAP11 and RIα are degraded by autophagy. (A) Brain lysates of Atg7F/F (Control) and Atg7F/F-SynCre (cKO) were subjected to immunoblotting analysis with the indicated antibodies. (B) Quantification of protein levels in A. Unpaired Student’s t tests were used, and values are presented as mean ± SEM (n = 3 mice/genotype). **P < 0.01; ***P < 0.001; ns, not significant. (C) Brain lysates of Atg14F/F (Control) and Atg14F/F-SynCre (cKO) were subjected to immunoblotting analysis with the indicated antibodies. (D) Quantification of protein levels in C. Unpaired Student’s t tests were used, and values are pre- sented as mean ± SEM (n = 4 mice/genotype). **P < 0.01; ***P < 0.001; ns, not significant. (E) Atg7 WT and Atg7 KO MEF cells were nutrient starved by using EBSS for the indicated times, and cells were assayed by immunoblotting analysis with the indicated antibodies. (F) Quantification of the protein levels from E was obtained by normalizing the level of proteins to Actin, respectively, and further to the respective control. One-way ANOVA was used in each genotype, and values are presented as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. (G) Atg7 WT and Atg7 KO HEK293T cells were nutrient starved by using EBSS for the indicated times and were then assayed by immunoblotting analysis with the indicated antibodies. (H) Quantification of the protein levels from G was obtained by normalizing the level of proteins to Actin, respectively, and further to the respective control. One-way ANOVA was used in each genotype, and values are presented as mean ± SEM (n = 4). #P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Control, Western Blot

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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 2. AKAP11 is an autophagy adaptor harboring LC3-interacting region. (A) Purified GST, GST-LC3A, GST-LC3B, or GST-GABARAP proteins were incubated with HEK293T lysates expressing HA-AKAP11 and subjected to pull-down assay. Interaction of HA-AKAP11 and GST-fusion proteins was detected by im- munoblotting analysis with the indicated antibodies. (B) HEK293T cells stably expressing GFP-LC3B were subjected to GFP immunoprecipitation. Interaction of AKAP11 and GFP-LC3B was detected by immunoblotting analysis with the indicated antibodies. (C) The alignment of LIR (LC3-interacting region) motif in AKAP11 from different species. (D) GST and GST-LC3B were subjected to pull down in HEK293T cellular lysates in the presence of HA-AKAP11-WT and mutLIR. Interaction of HA-AKAP11 and GST-LC3B was detected by immunoblotting analysis with the indicated antibodies. (E) HEK293T cells stably expressing GFP- LC3B were transfected with HA-AKAP11-WT or HA-AKAP11-mutLIR. Cellular lysates were subjected to GFP immunoprecipitation. Interaction of HA-AKAP11 and GFP-LC3B was detected by immunoblotting analysis with the indicated antibodies. (F) Quantification of the results from E was obtained by normalizing levels of immunoprecipitated HA-AKAP11 to the level of input and then further normalizing to the level of immunoprecipitated GFP-LC3B. Paired Student’s t tests were used, and values are presented as the mean ± SEM (n = 3). **P < 0.01. (G) HeLa cells transfected with HA-AKAP11 were subjected to nutrient starvation in the presence of Baf A1 and followed by immunostaining with anti-HA and anti-LC3 antibodies. Representative images are shown. (Scale bar, 10 μm.) (H and I) Quantification of the results from G. One-way ANOVA was used, and values are presented as mean ± SEM (more than 50 cells were calculated from three independent experiments). ***P < 0.001.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Purification, Incubation, Expressing, Pull Down Assay, Stable Transfection, Immunoprecipitation, Western Blot, Transfection, Immunostaining

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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 3. AKAP11 mediates RIα degradation. WT and AKAP11 KO HEK293T (A) and HeLa (C) cells were assayed by immunoblotting analysis with the indicated antibodies. (B and D) Quantification of the results from A and C were obtained by normalizing protein levels to Actin, respectively, and further to the re- spective control. Paired Student’s t tests were used, and values are presented as the mean ± SEM (A, n = 5; C, n = 4). *P < 0.05; **P < 0.01; ns, not significant. WT and AKAP11 KO HEK293T (E) and HeLa (G) cells were subjected to nutrient starvation by using EBSS, and cells were then assayed by immunoblotting analysis with the indicated antibodies. (F and H) Quantification of the results from E and G were obtained by normalizing protein levels to Actin, respectively, and further to the respective control. One-way ANOVA was used in each genotype, and values are presented as mean ± SEM (n = 3). * and #P < 0.05; ##P < 0.01; ###P < 0.001. (I) WT and AKAP11 KO HeLa cells were transfected with FLAG-RIα and subjected to nutrient starvation in the presence of Baf A1. Cells were then immunostained with anti-FLAG and anti-LC3 antibodies. (Scale bar, 10 μm.) (J and K) Quantification of the results from I. One-way ANOVA was used, and values are presented as the mean ± SEM (more than 40 cells were calculated from three independent experiments). ***P < 0.001. (L) WT and AKAP11 KO HEK293T cells were cotransfected with FLAG-LC3 construct, and either HA-AKAP11-WT or HA-AKAP11-mutLIR and were assayed by immunoprecipitation with anti-FLAG antibody. The interaction of FLAG-LC3 and HA-AKAP11 as well as FLAG-LC3 and RIα was detected with the indicated antibodies. (M) Quantification of the results from L was obtained by normalizing levels of immunoprecipitated HA-AKAP11 and RIα to the level of input and further to the level of immunoprecipitated FLAG. Paired Student’s t tests were used, and values are presented as the mean ± SEM (n = 3). **P < 0.01.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Western Blot, Control, Transfection, Construct, Immunoprecipitation

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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 4. Glucose deprivation causes AKAP11-dependent PKA activation. (A and B) WT and AKAP11 KO HEK293T cells were transfected with AKAR4 biosensor construct; at 24 h posttransfection, cells were subjected to glucose starvation (GS) for the indicated time and were then assayed by performing live imaging for 5 min at indicated time points. (A, Top) Representative images are shown. (Scale bar, 10 μm.) (Bottom) Scheme of AKAR4 biosensor. (B) Quantification of the results from A was obtained by calculating the ratio of Y (the intensity of cpVenus) and C (the intensity of Cerulean) and was then further normalized to each control. Two-way ANOVA was used, and values are presented as mean ± SEM (around 40 cells were calculated from three independent experiments). ***P < 0.001. (C) WT and AKAP11 KO HEK293T cells were subjected to GS for the indicated times and were then assayed by immunoblotting analysis with the indicated antibodies. (D) Quantification of the results from C was obtained by normalizing p-CREB to total CREB levels and further to the respective control. Two-way ANOVA was used, and values are presented as mean ± SEM (n = 7). *P < 0.05. (E) Quantification of the results from C was obtained by normalizing the level of proteins to Actin, respectively, and further to the respective control. One-way ANOVA was used for each genotype, and values are presented as mean ± SEM (n = 7). ** and ##P < 0.01.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Activation Assay, Transfection, Construct, Imaging, Control, Western Blot

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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 5. AKAP11 regulates PKA-mediated mitochondrial metabolism in response to glucose starvation (GS). (A and B) WT and AKAP11 KO HEK293T cells were treated with GS for 1 h and were then subjected to seahorse assay. One-way ANOVA was used. Data are presented as mean ± SEM (n = 10 biological replicates). **P < 0.01; ***P < 0.001; ns, not significant. (C and D) WT and AKAP11 KO HEK293T cells were transfected with AKAP11-WT or mutLIR constructs and subjected to seahorse assay with low glucose medium. One-way ANOVA was used. Data are presented as mean ± SEM (n = 9 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. (E and F) WT and AKAP11 KO HEK293T cells were treated with either DMSO or 20 μM forskolin (FSK) and 200 μM IBMX for 1 h and were then subjected to seahorse assay with low glucose medium. Data are presented as mean ± SEM (n = 5 biological replicates). *P < 0.05; **P < 0.01; ns, not significant. (G) WT and AKAP11 KO HEK293T cells were subjected to GS for the indicated times, and cells were then assayed by immunoblotting analysis with the indicated antibodies. (H) Quantification of the results from G was obtained by normalizing the level of proteins to Actin, respectively, and further to the respective control. Two-way ANOVA was used, and values are presented as mean ± SEM (n = 4). *P < 0.05; **P < 0.01.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Transfection, Construct, Western Blot, Control

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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 6. AKAP11 controls mitochondrial elongation and cell viability through PKA phosphorylation of DRP1 upon glucose starvation (GS). (A) WT and AKAP11 KO HeLa cells were subjected to GS for 1 h and immunostained with TOMM20 antibody. Representative images are shown. (Scale bar, 10 μm.) (B) Quanti- fication of the results from A. One-way ANOVA was used, and values are presented as mean ± SEM (more than 80 cells were calculated from three inde- pendent experiments). ***P < 0.001; ns, not significant. (C) WT and AKAP11 KO HeLa cells were subjected to GS for 1 h, and cells were then processed for EM imaging. Representative images are shown. (Scare bar, 1 μm.) (D) WT and AKAP11 KO HeLa cells were subjected to GS for the indicated times and were then assayed by immunoblotting analysis with the indicated antibodies. (E) Quantification of the results from D was obtained by normalizing the level of proteins to Actin, respectively, and further to the respective control. Two-way ANOVA was used, and values are presented as mean ± SEM (n = 5). *P < 0.05; **P < 0.01. (F) WT and AKAP11 KO HEK293T were subjected to GS for the indicated time and were then measured by cell viability assays. Two-way ANOVA was used, and data are presented as mean ± SEM (n = 3). **P < 0.01. (G) WT and AKAP11 KO HEK293T were either transfected with HA-AKAP11-WT or HA-AKAP11-mutLIR, then subjected to GS for 24 h, and measured by cell viability assays. One-way ANOVA was used. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (H) WT and AKAP11 KO HEK293T were transfected with scramble RNA and PRKAR1A siRNA, subjected to GS for 24 h, and measured by cell viability assays. Two-way ANOVA was used. Data are presented as mean ± SEM ***P < 0.001; ****P < 0.0001.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Phospho-proteomics, Imaging, Western Blot, Control, Transfection

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

Article Title: Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.

doi: 10.1073/pnas.2020215118

Figure Lengend Snippet: Fig. 7. AKAP11 is required for PKA activation and colony formation of tumor cells. (A) Five glioma cell lines (U251, U87, LN229, SF295, and SF763) and HeLa (WT and AKAP11 KO) cells were assayed by immunoblotting analysis with the indicated antibodies. (B) Quantification of the results from A was obtained by normalizing the level of AKAP11 to Actin. One-way ANOVA was used, and values are presented as mean ± SEM (n = 3). ***P < 0.001. (C) Control and shRNA- AKAP11 U87, U251, and SF763 cells were assayed by immunoblotting analysis with the indicated antibodies. (D) Quantification of the results from C was obtained by normalizing the indicated proteins level to Actin. Unpaired Student’s t tests were used, and values are presented as mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. (E) Control and shRNA-AKAP11 SF763 cells were treated with 20 mM 2-DG for 6 h and were then assayed by immunoblotting analysis with the indicated antibodies. (F) Quantification of the results from E was obtained by normalizing p-CREB to total CREB levels and further to the respective control. One-way ANOVA was used, and values are presented as mean ± SEM (n = 3). *P < 0.05; ns, not significant. (G) Control and shRNA-AKAP11 SF763 cells were treated with 20 mM 2-DG for 24 h and were then measured by cell death assays. Two-way ANOVA was used. Data are presented as mean ± SEM (n = 4). **P < 0.01. (H) The colony formation assay was performed in Control and shRNA-AKAP11 cells (U87 and SF763). The representative images were shown. (Scale bar, 5 mm.) (I) The colony formation efficiency in H was assessed by normalizing the number of colonies to that in the Control. Unpaired Student’s t tests were used. Data are presented as mean ± SEM (U87, n = 4; SF763, n = 6). **P < 0.01.

Article Snippet: To generate the stable Control and shAKAP11 knockdown cells, 90% confluent glioblastoma cells U87, U251, and SF763 were infected with lentiviral particles carrying control mixed shRNA against AKAP11 (shAKAP11) (Santa Cruz Biotechnology, control, sc-108080;AKAP11, sc-105049-V) and 10 μg/mL polybrene.

Techniques: Activation Assay, Western Blot, Control, shRNA, Colony Assay