Supplementary Figure 1

Transcription

1 Supplementary Figure 1 ZV 50 nm Relative to Protein Levels () C Relative to Protein Levels () Treatment Time (6 h) ZV Concentration (25 µm) ZV Concentration (25 µm)

2 Supplementary Figure 2 ZV 50 nm Treatment Time (6 h) Relative to Protein Levels () C Relative to Protein Levels () ZV Concentration (25 µm) ZV Concentration (25 µm)

3 Supplementary Figure 3 XP h (25 µm) h C XP-1 XP (25 µm) h / (25 µm)

4 Supplementary Figure 4 Non-differentiated 3T3-L μm Relative density Relative density (6 h) Differentiated 3T3-L μm Relative density Relative density (6 h)

5 C Differentiated SGS μm Relative density Relative density (6 h) D Differentiated 3T3-L1 Relative mrn Levels Concentration (μm)

6 Supplementary Figure 5 LC3- I LC3-II μm Relative density ctin 12.5 µm C 2,000 X 4,000 X 10,000 X

7 Supplementary Figure 6 No Tx 100 nm 25 µm / 7 Relative density Relative density ctin 1.5 Relative mrn levels (5) No Tx / C nm 25 µm Relative mrn levels (7) No Tx / 100 nm 25 µm

8 Supplementary Figure 1. Differential activation of the UPR by HIV PIs in nondifferentiated 3T3-L1 cells. Non-differentiated 3T3-L1 cells were treated for 6 hwith 25 µm of 9 HIV PIs. mprenavir (), Indinavir (), tazanavir (ZV), Ritonavir (), Lopinavir (), Nelfinavir (), Saquinavir (), Darunavir (DV), Tipranavir (). ) Representative immunoblots against, and from nuclear extracts are shown. -C) The density of immunoblots was determined by Image J. Relative protein levels of and were normalized using as loading control. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, p < 0.05 and p<0.01. Supplementary Figure 2. Differential activation of the UPR by HIV PIs in differentiated 3T3-L1 cells. Non-differentiated 3T3-L1 cells were treated for 6 h with 25 µm of 9 HIV PIs. mprenavir (), Indinavir (), tazanavir (ZV), Ritonavir (), Lopinavir (), Nelfinavir (), Saquinavir (), Darunavir (DV), Tipranavir (). ) Representative immunoblots against, and from nuclear extracts. -C) The density of immunoblots was determined by Image J. Relative protein levels of and were normalized using as loading control. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, p < 0.05 and p<0.01. Supplementary Figure 3. Time-dependent activation of the UPR in mouse adipocytes by HIV PIs. Representative immunoblots of five separate experiments against,, XP-1, and from the nuclear extracts of differentiated mouse 3T3-L1 cells treated with 25 µm of HIV PIs for 0-24 h. was used as loading control. ) Ritonavir (); ) Lopinavir (); C) Lopinavir/Ritonavir (/). The density of immunoblots was determined by Image J. Relative protein levels of and were normalized using as loading control. 1

9 Supplementary Figure 4. ctivation of the UPR by. ) Non-differentiated 3T3L1 cells, ) differentiated 3T3-L1 cells, and C) differentiated human SGS were treated with increasing concentrations of for 6 h. Representative immunoblots against and from nuclear extracts are shown. The density of immunoblots was determined by Image J. Relative protein levels of and were normalized using as a loading control. D) Differentiated 3T3-L1 cells were treated with increasing concentrations of for 4 h and total RN was isolated. The mrn levels of and were quantified by real-time RT-PCR and normalized using internal control β-ctin. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, p < Supplementary Figure 5. induces an increase of autophagosomes in 3T3-L1 cells. ) Representative immunoblots of LC3 from total cell lysates of differentiated 3T3-L1 cells treated with increasing concentrations of for 24 h are shown. β-actin was used as loading control. ) Representative fluorescent images of non-differentiated 3T3-L1 cells stably expressing GFP-tagged LC3 treated with 12.5 µm or vehicle control () for 24 h. C) Representative EM images of non-differentiated 3T3-L1s treated with individual 12.5 µm for 24 h. Cells were processed for transmission electron microscopy as described in Methods. Representative images at 2,000, 4,000 and 10,000 are shown. Supplemental Figure 6. Effect of HIV PIs on 5 and 7 expression in differentiated mouse adipocytes. ) Representative immunoblots against 7 and 5 from total cellular extracts of mouse differentiated 3T3-L1 cells treated with vehicle control (), 100 nm, or (25 µm), (25 µm), or / (4:1, =20 µm, =5µM), for 24 h are shown. The density of immunoblot was determined by Image J. Relative protein levels of 5 and 7 were normalized using β-ctin as a loading control. -C) Total cellular RN was isolated from differentiated 3T3-L1 cells treated as in 2

10 () for 24 h. The mrn levels of ) 5 and C) 7 were quantified by real-time RT- PCR and normalized using internal control β-ctin. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, p <