Fig 1.
Detection of SQSTM1/LC3B, SQSTM1/GL1, NIX/GL1 and NIX/LC3B interactions by P-LISA.
(A) MDA-MB-436 cells were cultured for 24 h at 37°C and 5% CO2, fixed, permeabilized, blocked with 5% BSA, incubated with rabbit anti-LC3B, rabbit anti-GL1, mouse anti-SQSTM1 or/and mouse anti-NIX antibodies overnight at 4°C and then with an Alexa Fluor 488 goat anti-rabbit and an Alexa Fluor 555 goat anti-mouse, respectively, for 1 h. The cells were then analyzed using a confocal microscope. (B) For P-LISA, the protocol was performed according to the manufacturer’s recommendations using the same antibodies as described above. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. Scale bars: 20μm.
Fig 2.
Technical controls demonstrating the specificity of P-LISA signals.
(A) MDA-MB-436 cells were cultured for 24 h at 37°C and 5% CO2. P-LISA were performed according to the manufacturer’s recommendations. No primary antibodies were added before performing P-LISA with PLA R+ (anti-rabbit) and PLA M- (anti-mouse) (left panel); P-LISA SQSTM1/LC3B was also performed with PLA R+ (anti-rabbit) against LC3B and with PLA G- (anti-goat) unable to recognize SQSMT1 (left panel). Similar controls were performed for P-LISA SQSTM1/GL1, P-LISA LC3B/NIX and P-LISA GL1/NIX. (B) Quantification of LC3B mRNA expression in MDA-MB-436 cells following LC3B siRNA transfection analyzed using qRT-PCR (top panel). Quantification of SQSTM1/LC3 interactions detected by P-LISA in MDA-MB-436 cells transfected or not with LC3B siRNA (bottom panel) according to the manufacturer’s recommendations using rabbit anti-LC3B and mouse anti-SQSTM1 antibodies. (C) Absence of SQSTM1/LC3B P-LISA signals in murine cells since the anti-SQSTM1 antibody is specific of the human SQSTM1 protein and restoration of P-LISA signals when these cells were transfected with a vector coding the human HA-SQSTM1 protein. (D) Absence of GL1 protein was validated using western blotting in MDA-MB-436 cells expressing or not a GABARAPL1 shRNA (E) Quantification of NIX/GL1 interactions was performed by P-LISA in MDA-MB-436 cells expressing or not a GABARAPL1 shRNA using rabbit anti-GL1 and mouse anti-NIX antibodies.
Fig 3.
Increase of SQSTM1/GL1 and NIX/GL1 interactions quantified by P-LISA in MCF-7 overexpressing FLAG-GABARAPL1-6His and GFP-NIX.
(A) Expression of GL1 protein was analyzed using western blotting in MCF-7 expressing or not FLAG-GL1-6His. (B) MCF-7 Control or MCF-7 FLAG-GL1-6His cells were cultured for 24 h at 37°C and 5% CO2, fixed, permeabilized, blocked with 5% BSA, incubated with mouse anti-SQSTM1 and rabbit anti-GL1 antibodies antibodies overnight at 4°C and then with an Alexa Fluor 555 goat anti-mouse and an Alexa Fluor 488 goat anti-rabbit, respectively, for 1 h. The cells were then analyzed using a confocal microscope (top panel). For P-LISA, the protocol was performed according to the manufacturer’s recommendations using mouse anti-SQSTM1 and rabbit anti-GL1 antibodies (bottom panel). (C) MCF-7 FLAG-GL1-6His cells were cultured for 24 h at 37°C and 5% CO2 and transfected with pEGFP-NIX plasmid and then fixed and permeabilized. P-LISA was performed as precognized by the manufacturer using rabbit anti-GL1 and mouse anti-NIX antibodies. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. Scale bar: 20μm.
Fig 4.
Co-localization of SQSTM1/LC3B, SQSTM1/GL1, NIX/LC3B and NIX/GL1 P-LISA signals with the GFP-LC3B protein.
MDA-MB-436 cells were transfected with the pEGFP-LC3B vector then cultured with EBSS and BafA1 (100 nM) for 2 h. (A) Co-localization of SQSTM1/LC3B P-LISA signal and GFP-LC3B fluorescence. (B) Co-localization of SQSTM1/GL1 P-LISA signal and GFP-LC3B fluorescence. (C) Co-localization of NIX/LC3B P-LISA signal and GFP-LC3B fluorescence. (D) Co-localization of NIX/GL1 P-LISA signal and GFP-LC3B fluorescence. Co-localization of P-LISA signal and GFP-LC3B puncta was determined in at least 30 cells using the imageJ software. Nuclei were stained with DAPI. ****: p≤0.0001 and ***: p≤0.001, vs control (n = 3). Scale bar: 20μm.
Fig 5.
Effects of Rapamycin, Rotenone and CCCP on ATG8/cargo adapter interactions.
MDA-MB436 cells were cultured for 24 h at 37°C and 5% CO2 then with Rapa (10 μM for 5h), Rot (50 μM for 24h) or CCCP (100 μM for 24h). (A) SQSTM1/LC3B, (B) SQSTM1/GL1, (C) NIX/LC3B and (D) NIX/GL1 P-LISA were performed according to the manufacturer’s recommendations using mouse anti-NIX or anti-SQSTM1 and rabbit anti-GL1 or anti-LC3B antibodies. Nuclei were stained with DAPI. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. (E) Quantification of P-LISA signals (dots/cell and intensity per dot) was performed using the Blobfinder software. Each bar (Mean ± SEM) represents the mean obtained from the quantification of signals observed in about 200 cells chosen randomly in 5 different fields from 3 independent experiments. ****: p≤0.0001, ***: p≤0.0001, and **: p≤0.001, vs control. Scale bar: 20μm.