SUPPLEMENTARY INFORMATION. Chemical modulation of Chaperone-mediated autophagy by novel

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1 SUPPLEMENTARY INFORMATION Chemical modulation of Chaperone-mediated autophagy by novel retinoic acid derivatives Jaime Anguiano 1, Thomas P Garner 2, Murugesan Mahalingam 1, Bhaskar C. Das 1, Evripidis Gavathiotis 2,3,4 and Ana Maria Cuervo 1,4,5* Inventory supplementary information Supplementary Results Supplementary Figures 1-21 Supplementary Table 1-2 1

2 Supplementary Results Supplementary Figure. 1. Macroautophagy in cells with and without RAR. (a,b) Rates of degradation of long-lived proteins in control and RAR knock-down cells maintained in presence (left) or absence (right) of serum and left untreated (a) or treated with ATRA (b). Values are expressed as percentage of proteolysis. (n 3). (c) Immunofluorescence for LC3 in the same cells untreated or after blockage of lysosomal degradation with NH4Cl and leupeptin. Left: representative images of cells in regular media. Insets show higher magnification. Right: quantification of changes in number of pucta per cell after lysosomal inhibition. Values are mean+s.e. (n 3). * p

3 Supplementary Figure. 2. Effect of knockdown of RAR on autophagic pathways. (a) Autophagic flux in mouse fibroblasts control (ctr) or knocked-down for RAR (RAR (-)) expressing mcherry-gfp- LC3 and maintained in the presence or absence of serum. Merged images are shown. The boxed areas at higher magnification are shown in Fig. 2d). (e) Control and RAR (-) cells were transfected with the KFERQ-mcherry1 photoactivable reporter and after photoactivation were maintained in media with or without serum. Representative images The boxed areas at higher magnification are shown in Fig. 2f.. 3

4 Supplementary Figure. 3. Effect of ATRA supplementation on autophagic pathways. (a) Autophagic flux in mouse fibroblasts untreated (None) or treated with (40 M) ATRA expressing mcherry-gfp-lc3 and maintained in the presence or absence of serum. Merged images are shown. The boxed areas at higher magnification are shown in Fig. 3d). (e) Control and RAR (-) cells were transfected with the KFERQ-mcherry1 photoactivable reporter and after photoactivation were maintained in media with or without serum. Representative images The boxed areas at higher magnification are shown in Fig. 2f. 4

5 Supplementary Figure. 4. Effect of ATRA supplementation on autophagy in cells deficient in RAR. (a) Rates of degradation of long-lived proteins in control and RAR knock-down cells maintained in presence or absence of serum and supplemented or not with ATRA. Values are expressed as percentage of proteolysis. (n 3). (b) CMA activity in the same cells expressing the KFERQ-mcherry-1 fotoactivatable reporter. Left: representative fields with higher magnification insets. Nucleus are highlighted by DAPI. Right: Average number of fluorescent puncta per cell quantified in >50 cells in at least 4 different fields. Values are mean+s.e. Differences with untreated samples (*) or with control cells ( ) are significant for * p

6 Supplementary Figure 5. Effect of retinoid derivatives on cellular viability. Mouse fibroblasts were treated with the indicated concentrations of the retinoid derivatives AR7, GR1 and GR2 for 12h. (a) Cellular viability expressed as percentage of the viability in untreated cells. Dotted lines mark the acceptable range of changes in cellular viability (IC80). (b) Cell dead measured in cells subjected to the indicated treatments in the absence of serum measured upon double staining with Annexin V and 7ADD and FACs analysis. Top, representative FACs plot for cells treated with 20 M of the retinoid derivatives. Bottom, percentage of cells negative for both staining. Cells treated with staurosporine at the indicated doses are used as positive control. 6

7 Supplementary Figure 6. Library of retinoid derivatives. (a) Examples of structures of three groups of retinoid derivatives generated in this study. (b) Examples of the effect of some of these compounds on CMA activity in mouse fibroblasts maintained in the presence of serum. A photoactivable KFERQ-PAmCherry1 reporter (see Material and Methods) was used in untreated (None) or cells treated with 20 M of the indicated compounds. Representative images are shown. Nuclei were highlighted with DAPI. 7

8 Figure 7. Doses-dependence of the effect of the retinoid derivatives on CMA activity. Mouse fibroblasts expressing the KFERQ-mcherry1 photoactivable reporter were treated with the indicated concentrations of GR1 and imaged 16 h after photoactivation. Rigth: higher magnification images. Nuclei are labeled with DAPI. Untreated cells and cells treated with 40 M ATRA are also shown on the right. Quantification of these experiments is shown in Fig. 5b. 8

9 Supplementary Figure 8. Purity and properties of the novel retinoid derivatives. (a) Synthetic reaction schemes for the two novel guanidine retinoids (GR1 and GR2) and the atypical retinoid (AR7). (b-d) 1 H-NMR, 13 C-NMR and mass spectra for the lead retinoid derivatives AR7 (b), GR1 (c) and GR2 (d). NMR revealed that GR1 compound had a mixture of isomers with E- and Z-stereoselectivity in 2:1 ratio and GR2 had a mixture of isomers with E- and Z-stereoselectivity in 1:0.2 ratio. 9

10 Supplementary Figure 9. Effect of novel retinoid derivates on RAR activity. Mouse fibroblasts were co-transfected with the hrar receptor construct, a relevant reporter luciferase plasmid and the nonretinoid regulated renilla reporter to control for transfection. Values show luciferase units detected in cells subjected to: (a) the indicated concentrations or 50 nm (b) of ATRA and three different groups of retinoid derivaties generated in this study for 12h. (c) 100nM ATRA alone (ATRA) or in the presence of 50nM of the retinoid derivatives. Values show luciferase intensity expressed as relative luminescence units (RLU). (n 3). The agonist AM580 and antagonist BMS614 are shown for comparative purposes. Values are mean+s.e. 10

11 Supplementary Figure 10. Effect of novel retinoid derivates on RXR activity. Mouse fibroblasts were co-transfected with the hrxr receptor construct, a relevant reporter luciferase plasmid and the non-retinoid regulated renilla reporter to control for transfection. Values show luciferase units detected in cells subjected to: (a) the indicated concentrations of ATRA and three different groups of retinoid derivaties generated in this study for 12h. (b) 10 M ATRA alone (ATRA) or in the presence of 5 M of the retinoid derivatives. Values show luciferase intensity expressed as relative luminescence units (RLU). (n 3). The antagonist BMS614 is shown for comparative purposes. Values are mean+s.e. 11

12 Supplementary Figure 11. Comparison of the novel retinoid derivatives with the RAR antagonist BMS614. (a) Effect of the three leading compounds and BMS614 on RAR and RCR activities when administered to mouse fibroblasts alone (left) or in the presence of ATRA at the indicated ratio analyzed used luciferase-based reporters. Values show luciferase intensity expressed as relative luminescence units (RLU). (n 3). (b) Mouse fibroblasts were transfected with the KFERQ-mcherry1 photoactivable reporter and supplemented or not with the indicated compounds (10 M). Left, representative fields and high magnification insets. Nuclei are highlighted with DAPI. Right, average number of fluorescent puncta per cell quantified in >50 cells in at least 4 different fields. Differences with untreated (*) are significant for p<0.01. (c) Immunoblot for LC3 of cells maintained in the presence or absence of serum and treated with 20 M of AR7 or BMS614 and protease inhibitors (PI), as labeled. Actin is shown as loading control. Bottom, increase in levels of LC3-II after PI treatment (LC3-II flux) calculated from the densitometric quantification of immunoblots. Percentage of inhibition is shown. All values are mean+s.e. 12

13 Supplementary Figure 12. Effect of retinoid derivatives on intracellular protein degradation. Rates of degradation of long-lived proteins in control (a) and RAR knock-down (-) cells (b) maintained in the presence of serum alone (None) or in the presence of 20 M of the indicated retinoid derivatives. Values are expressed as percentage of proteolysis and are mean +S.E. (n=3) 13

14 Supplementary Figure. 13. Characterization of the effect of the retinoid derivatives on CMA. Mouse fibroblasts control (Ctr) knocked-down (-) for RAR a LAMP-2A b or LAMP-2B c were transfected with the KFERQ-mcherry1 photoactivable reporter with or without the indicated compounds (20 M). Representative fields and high magnification insets are shown. Bar: 10 m. Nuclei are labeled with DAPI. Quantification of these experiments is shown in Fig. 6 c,d. 14

15 Supplementary Figure 14. Molecular dynamics simulations starting from docking pose I (energetically more favorable) and docking pose II of (a) AR7, (b) GR1 and (c) GR2 compounds in the binding pocket of RARα. Ribbon representations of the binding pocket overlaying the starting pose (blue sticks) calculated by docking and final pose (red sticks) generated by the molecular dynamics simulations are shown for each compound. Plots demonstrate the distance fluctuations over the time course of the molecular dynamics simulations for each docking pose. Two distances are selected from each pose to best represent the fluctuations of the compounds rather the protein during the molecular dynamics simulations. Selected distances correspond to carbon atoms at the center and the edge of each molecule and carbon atoms of L266 (green line) and T233 (blue line) for pose I and F228 (Magenta line) and S287 (red line) for pose II. 15

16 Supplementary Figure 15. Molecular docking of the E- and Z-isomers of GR1 and GR2 in the RARα binding pocket. Ribbon representation of the RARα binding pocket overlaying the E-isomer (blue sticks) and Z-isomer (red sticks) of GR1 and GR2 compounds in docking pose I and II. Both isomers of GR1 and GR2 have the lowest energy structures in docking pose I. Table shows the docking scores for the three leading molecules including the two isomers of GR1 and GR2. 16

17 Supplementary Figure 16. Superposition of the crystal structures of RARα in active (red) and inactive (grey) conformation and docked structures of AR7, GR1 and GR2. Compounds docked in pose I form steric clashes with the closed and active conformation of α-helix H12 but not with the open one. As a result, this docked orientation for AR7, GR1 and GR2 compounds is consistent with the proposed antagonistic mechanism that stabilizes α-helix H12 in an open and inactive conformation. 17

18 Supplementary Figure. 17. Effect of the retinoid derivatives in the oxidative cellular status and in lysosomes. (a) Oxyblot of cells untreated (none) or treated for 12 h with 20 M of the indicated compounds or 1 mm PQ. Actin is shown as loading control. (b) Rat liver lysosomes treated or not with protease inhibitors (PI) were incubated with GAPDH alone (None) or in the presence of 20 M of the indicated compounds. Immunoblot of samples collected by centrifugation (top) and quantification of the amount of GAPDH bound and taken up by each group of lysosomes (bottom). (n 3). 18

19 Supplementary Figure 18. Effect of the novel retinoid derivatives on the subcelluar distribution of the RAR. Mouse fibroblasts untreated or treated with (20 M) of ATRA and/or AR7 for 12 h were subjected to immunostaining for the RAR Top, representative fields with merged (left) or not (right) nuclear DAPI staining. Insets show higher magnification areas. Bottom, quantification of the fraction of cellular RAR detected in the nuclear in >25 cells in at least 4 different fields. Values are expressed as percentage. Differences with untreated samples (*) are significant for p Values are mean+s.e. 19

20 Supplementary Figure 19. Transcriptional dependence of the effect of the novel retinoid derivatives on CMA. (a) Mouse fibroblasts were transfected with the KFERQ-mcherry1 photoactivable reporter and treated with AR7 in the presence or absence of Actinomycin D. Left, representative fields and high magnification insets. Nuclei are highlighted with DAPI. Right, average number of fluorescent puncta per cell quantified in >50 cells in at least 4 different fields. (b) mrna levels of LAMP-1 in mouse fibroblasts control (Ctr) or knocked-down (-) for RAR and subjected to the indicated treatments. Values are corrected for actin and are expressed as folds control untreated cells. (n=3-4). (c) mrna levels of LAMP-2A in mouse fibroblasts treated or not with AR7 in the presence or absence of Actinomycin (n=4). (d) mrna levels of LAMP-2A in mouse fibroblasts maintained in presence or absence of serum and treated with AR7 or ATRA (n=3). Paraquat response is shown as positive control. Values are mean+s.e. Differences with untreated control (*) or between actinomycin treated or not ( ) are significant for * p<

21 Supplementary Figure 20. Effect of DNA load on cellular viability. Mouse fibroblasts were transfected with the indicated concentrations of an empty plasmid and left untreated or treated with 1mM PQ alone or in the presence of 20 M AR7. Viability expressed as folds that of untreated untransfected cells is shown. Values are mean+s.e. (n=3). 21

22 Supplementary Figure 21. Uncropped gels from main figures. Stars (*) denote bands immunoreactive to other proteins not relevant for this study or non-specific bands. 22

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