EMBO/soda-nel huggingface dataset:
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf6

(a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity-purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies against Tom1/Tom1L2. (b) Binding of myosin VI to Tom1 requires the WWY motif. Bottom, the mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild-type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n = 2 independent experiments. Top, Tom1 domain structure and truncation mutants. VHS, Vps-27, Hrs and STAM; GAT, GGA and Tom1. (c) RPE cells stably expressing GFP–myosin VI were processed for immunofluorescence microscopy to evaluate GFP–myosin VI and endogenous Tom1/Tom1L2 or Rab5 co-localization. The insets show higher magnifications of the areas outlined in the main panels. The arrows indicate areas of co-localization. (d) RPE cells with stable expression of GFP–myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP–myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP–myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP–myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP–myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf7

(a) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h. Western blot analysis was performed on whole-cell lysates against the indicated proteins. (b) Quantification of LC3-II intensity of western blots was performed by an infrared imaging system. (±s.d.; n = 3). *P0.05, **P0.01. (c, left) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). The insets show higher magnifications of the areas outlined in the main images. Nuclei are labelled with Hoechst (blue). (c,right,d) Quantification of LC3-positive punctae (c, right) and p62-positive punctae (d) was performed in a 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). The results are represented as the average punctae fluorescence intensity per cell (±s.d.) from n = 3 independent experiments each performed in triplicate wells, each with >500 cells. (e) HeLa cells with stable expression of the RFP–GFP–LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown (left). Confocal immunofluorescence microscopy (middle) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin (right). The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson’s coefficient of GFP/RFP overlap (±s.d.). A higher Pearson’s coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes. (f) Confocal immunofluorescence microscopy of Tom1-depleted RPE cells stably expressing GFP–LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images. (g) HeLa cells with stable expression of HttQ72–GFP were transiently transfected with Tom1 siRNA (top right), saponin-extracted, and processed for immunofluorescence microscopy (left) to quantify HttQ72–GFP punctae (bottom right). Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labelled with Hoechst. The results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots per cell (±s.d.; n = 3). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome (2012)
figf8

(a) Left, RPE cells with stable expression of GFP–LC3 following mock- or myosin-VI-siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labelled with Hoechst. The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co-localization and arrowheads highlight adjacent localization of Tom1 relative to LC3. Right, quantification was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. The results are represented as the average number of Tom1-positive vesicles per cell categorized by their relative position to LC3 (±s.d.; n = 3). (b) GFP–LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red–dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander’s overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum. (d) RPE cells stably expressing GFP–LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-red (TR)–dextran for 16 h followed by a chase period of 4 h. Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP–LC3 and 5 nm gold particles against Texas-red–dextran. Scale bars, 20 μm (a,b); 200 nm (d).
Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability (2019)
Figure 1

Figure 1. Degradation of MAGE-A3/6 proteins upon nutrient deprivation.

A, B MAGE-A3/6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times. Note both bands are MAGE-A3/6. Quantitation represents average of n=3 with standard deviation shown.

C Quantitative TMT proteomics of A375 cells grown in complete media (CM) or EBSS for 3 hrs. Number of proteins affected are shown. Autophagy related proteins are indicated in grey and MAGE-A proteins in blue.

D, E MAGE-A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 µg/mL translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n=3 with standard deviation shown.

Data information: Asterisks indicate ** p<0.01. ns indicates p>0.05 using 2-way ANOVA.
Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability (2019)
Figure 2

Figure 2. MAGE-A3/6 proteins are stabilized by insulin in FBS.

A, B FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown.

C Removal of FBS from normal growth conditions (DMEM+10% FBS) results in MAGE-A3/6 protein degradation in HeLa cells.

D, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown.

F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL.

G, H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown.

Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.
Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability (2019)
Figure 3

Figure 3. MAGE-A3/6 is degraded by the proteasome upon nutrient deprivation.

A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown.

B, C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown.

D Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.

E MAGE-A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times.

F, G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown.

H Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant negative (D/N) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment.

I Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 hrs after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.

Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.
Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability (2019)
Figure 4

Figure 4. The CRL4-DCAF12 E3 ubiquitin ligase degrades MAGE-A3/6.

A Tandem affinity purification of MAGE-A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage is shown.

B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP.

C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP.

D, E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5, or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).

F Knockdown of DCAF12 with individual siRNAs blocks MAGE-A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.

G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.
Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability (2019)
Figure 5

Figure 5. Specific MAGE-A proteins are regulated by the nutrient sensitive CRL4-DCAF12 ligase.

A Identification of DCAF12 substrates. Control (WT) or DCAF12 knockout (KO) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets. MAGE-A proteins (shown in blue) are stabilized in DCAF12 knockout cells.

B Knockout of DCAF12 rescues degradation of MAGE-A proteins in A375 cells. DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics. Note MAGE-A genes are not significantly altered by EBSS in DCAF12 KO cells.

C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome. DCAF12 targets (n=33) identified in (A) show significantly greater decrease upon EBSS treatment compared to non-DCAF12 altered genes (n=8243). Average with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).

D MAGE-A2, MAGE-A3, MAGE-A6, and MAGE-A12, but not other MAGE-A genes contain a DCAF12 degron motif (C-term -EE*).

E Overexpression of DCAF12 decreases wild-type MAGE-A3, but not MAGE-A3 degron mutant. The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting. The MAGE-A3 degron mutant was created as previously described [42], in which DNYNEPKANQ* were added to the extreme C-term to obscure the DCAF12 degron motif.

F Analysis of proteomics data (A-B) indicates that MAGE-A1, MAGE-A4, MAGE-A10, and MAGE-A11 are not regulated by nutrient deprivation or DCAF12. Average (n=3) with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).

G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.
Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability (2019)
Figure 6

Figure 6. Degradation of MAGE-A3/6 by DCAF12 is important for autophagy induction upon nutrient deprivation.

A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm.

C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm.

E A375 parental or DCAF12 KO cells were treated as described in (C). Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.

Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.
FMRP recruitment of β-catenin to the translation pre-initiation complex (2018)
Figure 3

Figure 3: β-catenin interaction with FMRP. A. (Left panel) A10 cells were transfected with Flag-FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β-catenin (green), Flag (FMRP, red), and nucleus (blue). (Bottom right panel) A line was drawn through a representative cell to indicate relative intensity of RGB signals. (Top right panel) An orthogonal projection was generated by a Z-stack (240 nm interval) image set using the ZEN program (Zeiss) depicting the region surrounding the nucleus in the XY, XZ, and YZ planes. B. (i) HEK 293T cells were transfected with Flag-FMRP as in A, and endogenous β-catenin protein complexes were immunoprecipitated with β-catenin antibody. Co-immunoprecipitated Flag-FMRP protein was detected by Western blot analysis with the Flag antibody. Non-programmed rabbit IgG was used as a control. (ii) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β-catenin or FMRP antibody. The corresponding protein was detected in the precipitated immunocomplex by Western blot. Non-programmed rabbit IgG was used as a control. C. (i) Purified 6x-His-FMRP and various regions of β-catenin proteins fused to GST were incubated with glutathione-agarose beads. Purified GST or GST-fusion proteins were visualized by Western blotting using the GST antibody, and sizes of corresponding GST-fusion proteins were confirmed. Pulled-down FMRP proteins by GST fused β-catenin were detected by immunoblotting with the FMRP antibody. (ii) Interaction between FMRP and β-catenin was assessed in the presence or absence of RNA. HEK 293T cells were subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody, in the presence or absence of RNase A (10 µg/mL). The corresponding protein was detected in the precipitated immunocomplex by Western blot, and non-programmed rabbit IgG was used as a control. RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A. D. (Top panel) A10 cells were transfected with GBP-Lifeact and either EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red) E. A10 cells were transfected as in (D) with GBP-Lamin B1.
FMRP recruitment of β-catenin to the translation pre-initiation complex (2018)
Figure 4

Figure 4: β-catenin and FMRP are co-localized in the pre-initiation complex. A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input. B. (i) eIF4E preferentially binds to the 5'cap (m7GTP) of mRNAs and recruits the pre-initiation complex to that site. (ii) m7GTP-agarose beads. C. (i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control. D. (i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C). (ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C). F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.
FMRP recruitment of β-catenin to the translation pre-initiation complex (2018)
Figure 5

Figure 5: β-catenin inhibits translation. A. Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error bars. B. A10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatment. C. Schematic depiction of rapamycin mediated inhibition of cap-dependent translation. D. A10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibody. E. A10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.