A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling

Transcription

1 SUPPLEMENTARY INFRMATIN A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling Rahul S. Kathayat 1, Pablo D. Elvira 1, Bryan C. Dickinson 1 * 1 Department of Chemistry, The University of Chicago, Chicago, IL *Corresponding author. address: Dickinson@uchicago.edu (Bryan C. Dickinson) Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-1

2 SUPPLEMENTARY RESULTS Supplementary Figure 1: UV-Vis spectra of DPP-1, DPP-2, and DPP µm of each, DPP-1 (a), DPP-2 (b), DPP-3 (c), and 5 µm of N,N-dimethylrhodol (d) in HEPES (20 mm, ph 7.4, 150 mm NaCl, 1% Triton X-100). DPP-1, DPP-2, and DPP-3 all show a major UV-Vis absorbance peak at 310 nm with extinction coefficients 6.3x10 3 M -1 cm -1 (DPP-1), 5.9x10 3 M -1 cm -1 (DPP-2), 3.7x10 3 M -1 cm -1 (DPP-3). The absorbance band at ~500 nm is the open, carboxylic acid form of the molecules. Carbamate formation on the phenol of N,Ndimethylrhodol in DPP-1, DPP-2, and DPP-3 dramatically shifts the equilibrium toward the closed, lactone form of the molecules, as indicated by the dramatic decrease in absorbance at ~500 nm. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-2

3 Supplementary Figure 2: SDS-PAGE gel of purified enzymes. Lane 1: protein ladder (from top to bottom 180, 130, 95, 72, 55, 43, 34, 26, 17 and 10 kda), lane 2: purified APT1, lane 3: purified APT2, lane 4: purified APT1(S119A), lane 5: purified APT2(S122A). Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-3

4 Supplementary Figure 3: Emission spectra of DPP-1, DPP-2, and DPP-3. Fluorescence emission spectra of (a) 5 µm DPP-1, (b) 5 µm DPP-2, or (c) 5 µm DPP-3 in HEPES (20 mm, ph 7.4, 150 mm NaCl, 0.1% Triton X-100), and emission spectra after treatment with either 50 nm APT1 or 50 nm APT2 for 20 min (λ ex = 485 nm). Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-4

5 Supplementary Figure 4: In vitro assays of DPP-2 and DPP-3 without detergent. (a) 25 µm DPP-2 in HEPES (20 mm, ph = 7.4, 150 mm NaCl), with either carrier control or 50 nm purified APT1 (λ ex 490/9 nm, λ em 545/20 nm). Error bars are ± standard deviation (n = 3). (b) 25 µm DPP-3 in HEPES (20 mm, ph = 7.4, 150 mm NaCl), with either carrier control or 50 nm purified APT1 (λ ex 490/9 nm, λ em 545/20 nm). Error bars are ± standard deviation (n = 3). Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-5

6 Supplementary Figure 5: In vitro assays with mutant enzymes. In vitro fluorescence assays of 5 µm DPP-1 (a), DPP-2 (b), or DPP-3 (c) in HEPES (20 mm, ph 7.4, 150 mm NaCl, 0.1% Triton X-100) with either 500 nm purified APT1(S119A) or APT2(S122A) (λ ex 490/9 nm, λ em 545/20 nm). Error bars are ± standard deviation (n = 3). Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-6

7 Supplementary Figure 6: Small molecule inhibitors block DPP-3 signal in vitro. Quantification of fluorescence response of 5 µm DPP-3 treated with either 50 nm purified APT1 or APT2 for 30 min each in the presence or absence of 50 µm of either PalmB or ML348 ( 348 ) (λ ex 490/9 nm, λ em 545/20 nm). Error bars are ± standard deviation (n = 4). Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-7

8 Supplementary Figure 7: Complete imaging series from Fig. 2a. HEK293T cells loaded with 1 µm DPP-2 for 15 min and then analyzed by fluorescence microscopy. Images for brightfield, Hoechst nuclear stain, and DPP-2 fluorescence signal are shown for each set of conditions. 50 µm scale bar shown. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-8

9 Supplementary Figure 8: Quantification of DPP-2 HEK293T cell imaging. Experiment shown in Fig. 2a and Supplementary Fig. 7. Error bars are ± standard deviation (n = 6). Statistical analysis performed with a two-tailed Student's t-test with unequal variance. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-9

10 Supplementary Figure 9: Analysis of DPP-2 inhibition by PalmB, ML348, and ML349 in live cells. HEK293T cells loaded with 5 µm DPP-2 for 5 min after treatment with either DMS, PalmB, ML348, or ML349, and then analyzed by flow cytometry. The data are displayed as two separate histograms to allow comparison of either the effects of ML348 (a) or ML349 (b). Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-10

11 Supplementary Figure 10: Complete imaging series from Fig. 2b. HEK293T cells loaded with 1 µm DPP-3 for 15 min and then analyzed by fluorescence microscopy. Images for brightfield, Hoechst nuclear stain, and DPP-3 fluorescence signal are shown for each set of conditions. 50 µm scale bar shown. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-11

12 Supplementary Figure 11: Quantification of DPP-3 HEK293T cell imaging. Experiment shown in Fig. 2b and Supplementary Fig. 10. Error bars are ± standard deviation (n = 6). Statistical analyses performed with a two-tailed Student's t-test with unequal variance. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-12

13 Supplementary Figure 12: DPP-2 vs DPP-3. HEK293T cells loaded with 1 µm DPP-2 or DPP- 3 for 15 m and then analyzed by fluorescence microscopy. Images for brightfield, Hoechst nuclear stain, and DPP-2/3 fluorescence signal are shown for each set of conditions. Microscope settings and processing identical for DPP-2 and DPP-3, showing substantially more signal arising from DPP-2, likely due to more efficient uptake. 50 µm scale bar shown. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-13

14 Supplementary Figure 13: RNAi knockdown of APT1. (a) Complete imaging series from Fig. 2d. After treatment with either sirna(control) or sirna(apt1), HEK293T cells loaded with 5 µm DPP-3 for 15 min and then analyzed by fluorescence microscopy. Images for brightfield, Hoechst nuclear stain, and DPP-3 fluorescence signal are shown for each set of conditions. (b) Quantification of experiment shown in (a). Error bars are ± standard error (n = 6). Statistical analysis performed with a two-tailed Student's t-test with unequal variance. (c) After treatment with either sirna (control) or sirna targeting APT1, HEK293T cells loaded with 5 µm DPP-3 for 5 min and then analyzed by flow cytometry. 50 µm scale bar shown. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-14

15 Supplementary Figure 14: Flow cytometry analysis of DPP-3 in HeLa, MCF-7, and MDA- MB-231 cells. HeLa (a), MCF-7 (b), or MDA-MB-231 (c) cells loaded with 5 µm DPP-3 for 5 min and then analyzed by flow cytometry. Inhibition by 1 µm PalmB or 1 µm ML348 knocks down the depalmitoylation signal as measured by DPP-3. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-15

16 Supplementary Figure 15: Effect of serum starvation on A431 cells. (a) Complete imaging series from Fig. 2e. Serum starved A431 cells or cells grown under normal growth conditions loaded with 5 µm DPP-3 for 15 min then analyzed by fluorescence microscopy. Images for brightfield, Hoechst nuclear stain, and DPP-3 fluorescence signal are shown for each set of conditions. (b) Serum starved or non-starved A431 cells loaded with 5 µm DPP-3 for 5 min and then analyzed by flow cytometry. Flow cytometry confirms that serum starvation increases the depalmitoylase activity as measured by DPP-3, but is knocked down to comparable levels by treatment with 1 µm PalmB or 1 µm ML µm scale bar shown. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-16

17 Supplementary Figure 16: EGF stimulation in serum starved A431 cells. (a) Complete imaging series from Fig. 2g. Serum starved A431 cells loaded with 5 µm DPP-3 for 15 min then analyzed by fluorescence microscopy in either the absence (control) or presence of 1 ng/ml EGF. Images for brightfield, Hoechst nuclear stain, and DPP-3 fluorescence signal are shown for each set of conditions. (b-d) Quantification of images from three separate biological replicate experiments of experiment shown in (a). Error bars are ± standard error (n = 5). Statistical analyses performed with a two-tailed Student's t-test with unequal variance. 50 µm scale bar shown. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-17

18 N N S-thiosulfinate S S N H R 2 R 1 N N N S sulfenyl-amide R 1 N N S-sulhydration S SH N H R 1 N N SH N H R 1 N N N N H S H R 1 sulfinic acid N N S-Nitrosothiol S N N H R 1 N N S-glutathionylation S S N H R 1 Glutathione Supplementary Figure 17: Generality of approach to study erasers of cysteine posttranslational modifications. As the catalog of known cysteine PTMs continues to expand, there is a need for tools to study the regulation and biological importance of each modification. Although in this work we focused our attention on one PTM, S-palmitoylation, in principle, the strategy outlined here can be immediately deployable to create fluorescent probes to monitor the erasers of any cysteine PTM. Dickinson et. al;; A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling, SI-18