Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: MAGED2 depletion induces autophagy under hypoxic conditions. HEK293 cells were transfected with control or MAGED2 siRNAs. Upon confluency 24–48 h post transfection, cells were exposed to physical hypoxia overnight. Total cell lysates were separated by SDS-PAGE and blotted for p62, ATGs and LC3B detection. Blotting for HIF-1α- was carried out to confirm its induction under hypoxia. Immunocytochemistry was carried out in parallel in HEK293 cells transfected and stressed similarly prior to incubation with LC3B antibody. ( A ) Representative Western blot images demonstrate decreased p62 levels, elevated ATG5-ATG12 complex levels and a higher LC3II abundance upon MAGED2 depletion in the presence of physical hypoxia. Leupeptin assay confirmed the induced autophagy where the highest LC3II accumulation corresponded to cells where MAGED2 is knocked down. ( B ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II from the immunoblot A. All samples shown on individual blots are from the same experiment and each blot represents an example of three independent experiments. ( C ) Representative immunofluorescence images displaying LC3B staining in control and MAGED2-transfected HEK293 cells under physical hypoxia. Scale bar 5 µm. ( D ) This notion was further supported by qRT-PCR, where the quantity of ATG5 and ATG12 was determined in HEK293 cells transfected with control or MAGED2 siRNAs and exposed to hypoxia. Total mRNA was isolated, and the relative mRNA amounts of both genes were measured. Statistical significance was determined by unpaired Student’s t -test. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Incubation, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Isolation

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Cobalt chloride induces autophagy in MAGED2-depleted HEK293 cells. HEK293 cells were transfected with control or MAGED2 siRNAs. Upon confluency 24–48 h post transfection, cells were treated with cobalt chloride (“chemical hypoxia”, CoCl 2 , 300 µM) for 14–16 h. Total cell lysates were separated by SDS-PAGE and blotted for p62, ATGs and LC3B detection. Of note, HIF-1α immunoblotting confirmed the hypoxic condition. Immunocytochemistry was carried out in parallel in HEK293 cells transfected and stressed similarly prior to incubation with LC3B antibody. ( A ) Representative Western blot images from HEK293 cells demonstrate reduced p62 levels, increased ATG5-ATG12 conjugate levels and a higher LC3II prevalence upon MAGED2 depletion. Coincubation with leupeptin (100 µM) led to an increased LC3II accumulation, which confirmed induction of autophagy. ( B ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II from immunoblot A. All samples shown on individual blots are from the same experiment, and each blot represents an example of three independent experiments. Bar graphs show mean ± SEM, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Representative immunofluorescence images displaying LC3B staining in control and MAGED2-transfected HEK293 cells treated with CoCl 2 . Scale bar 5 μM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Western Blot, Immunocytochemistry, Incubation, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Autophagy is significantly induced by classical ER-stressors in MAGED2-depleted cells. Control or MAGED2 siRNAs were transfected into HEK293 cells. At 24–48 h post-transfection, cells were treated with the ER stressors 600 nM tunicamycin (Tun) overnight or 10 μM brefeldin A (BFA) for 2 h. SDS-PAGE was used to separate total cell lysates before blotting for p62, LC3B and ATGs detection. Immunocytochemistry was conducted in parallel, and HEK293 cells were stained for the accumulation of LC3B puncta after being transfected and treated with ER stressors. Representative Western blot images from HEK293 cells treated with tunicamycin ( A ) or BFA ( D ) show reduced p62 levels combined with increased levels of ATG5-ATG12 complex and higher LC3II abundance in MAGED2-depleted cells. Leupeptin treatment led to the highest LC3II accumulation because of blocked autophagic flux. ( B , E ) Densitometric analysis of p62, ATG5-ATG12 conjugate and LC3II of the immunoblots ( A , D ) respectively. All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C , F ) LC3B staining in control and MAGED2-transfected HEK 293 cells treated with tunicamycin or BFA, respectively. The scale bar is 5 μM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Genotoxic stress enhances autophagy in MAGED2-depleted cells. HEK293 cells were transfected with control or MAGED2 siRNAs. Cells were treated 24–48 h post-transfection with 10 µM camptothecin (CPT) overnight. Total cell proteins were separated using SDS-PAGE and then immunoblotted for p62, ATGs and LC3B detection. In parallel, immunocytochemistry for HEK293 cells was carried out to stain for LC3B puncta following the same protocol. ( A ) Representative Western blot images from HEK293 cells treated with CPT show decreased p62 expression, increased levels of ATG5-ATG12 complex and higher LC3II abundance upon MAGED2 depletion. Treatment with leupeptin blocked the autophagic flux and resulted in the highest LC3II accumulation when MAGED2 was depleted. ( B ) Densitometric analysis of p62, ATG5-ATG12 complex and LC3II from the immunoblots in ( A ). All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Representative immunofluorescence images showing LC3B staining in control and MAGED2-transfected HEK293 cells upon CPT treatment. Scale bar 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Staining, Western Blot, Expressing, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Nutritional stress promotes autophagy in MAGED2-depleted cells. Control or MAGED2 siRNAs were transfected into HEK293 cells. Cells were treated 24–48 h post transfection with 4 mM 2-Deoxy-D-glucose (2DG) for 30 min. SDS-PAGE was used to separate total cell lysates, which were next blotted for p62, ATGs and LC3B detection. Immunocytochemistry was conducted as mentioned before, and both HeLa and HEK293 cells were stained for the accumulation of LC3B puncta. ( A ) Representative Western blot images from HEK293 cells treated with 2DG shows decreased p62 levels, elevated expression of ATG5-ATG12 complex, higher LC3II abundance upon MAGED2 depletion and the highest ratio when co-incubating with Leupeptin. ( B ) Densitometric analysis of P62, ATGs and LC3II from the immunoblots in ( A ). All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01. ( C ) Representative immunofluorescence images showing LC3B staining in control and MAGED2-transfected HEK293 cells upon 2DG treatment. Scale bar 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Staining, Western Blot, Expressing, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: GNAS depletion induces autophagy under hypoxic conditions. Control or GNAS siRNAs were transfected into HEK293 cells. Upon confluency, 24–48 h post transfection, hypoxic stress was applied overnight for one set in a modular chamber while the other set was kept in normoxic conditions. Cells were then lysed and blotted for detection of p62, LC3B and autophagy-related genes. HIF-1α immunoblot confirmed hypoxia. ( A ) A representative Western blot from HEK293 cells demonstrates that GNAS depletion induced autophagy significantly under hypoxia where the low p62 levels and ATGs upregulation were accompanied by a higher conversion to the lipidated form LC3II under hypoxia. ( B ) Densitometric analysis of p62, ATG5-ATG12 complex and the LC3II from the immunoblot A. All samples shown on individual blots are from the same experiment, and each blot represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( C ) Immunocytochemistry for HEK293 cells transfected with either control, MAGED2 or GNAS siRNAs and exposed to physical hypoxia for 14–16 h show the accumulation of LC3B puncta. Similar to MAGED2-depletion, knockdown of GNAS also led to an increased abundance of LC3B positive puncta. Scale bar is 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, Western Blot, Immunocytochemistry

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Forskolin reverses the induction of autophagy under stress conditions upon MAGED2 depletion. HeLa and HEK293 cells were both transfected with control and MAGED2 siRNA. After 24 to 48 h upon confluency, the media was changed to DMEM as control or DMEM containing 10 µM FSK before subjecting all cells to physical hypoxia overnight. SDS-PAGE was used to separate total cell proteins, which were further immunoblotted for LC3B detection. Hypoxic condition was verified by blotting for HIF-1α. Moreover, immunocytochemistry for HEK293 cells transfected with either control or MAGED2 siRNA and exposed upon confluency to overnight physical hypoxia with or without the addition of 10 µM of FSK was performed and the accumulation of LC3B puncta was analyzed. ( A , B ) Representative Western blot images from ( A ) Hela and ( B ) HEK293 cells treated with 10 µM FSK show a reduction in LC3B expression and a diminished autophagy induction upon FSK treatment. The promoted autophagy seen when MAGED2 is knocked-down is rendered to control levels by FSK addition. ( C , D ) Densitometric analysis of LC3B immunoblots in ( A , B ), respectively. All blots are from the same experiment, and each represents an example of three independent experiments. Bar graphs show mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( E ) Immunocytochemistry confirms that FSK addition to HEK293 cells prior to hypoxic stress reversed the observed autophagic induction, and the accumulation of puncta was rendered to control levels. Scale bar is 5 µM.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, SDS Page, Immunocytochemistry, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Proposed model for the role of MAGED2 under stress conditions (created with BioRender.com). Under stress, MAGED2 inhibits MDM2-dependent ubiquitination and endocytosis of Gαs. This ensures activation of the adenylate cyclase and cAMP generation and activation of PKA under stress. Reduced PKA activity impairs regulation of autophagy mediated by the cAMP/PKA pathway.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Activation Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway

doi: 10.3390/ijms241713433

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Anti-MAGED2 rabbit raised against this peptide (QVQENQDTRPKVKAK) , Eurogentec (Cologne, Germany) , .

Techniques: Transfection, Lysis, SYBR Green Assay, Real-time Polymerase Chain Reaction, Software