Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging -- Yang et al. 103 (37): 13872 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Yang et al. 10.1073/pnas.0601455103.

Supporting Information

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Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Text
Supporting Figure 10

Supporting Figure 7

Fig. 7. Spectral characterization of the folate-FRET reporter. (A) HPLC chromatographs (Left) and the corresponding absorption spectra (Right) for the folate-FRET reporter before (Upper) and after (Lower) disulfide reduction. (B) The emission spectra of the folate-FRET reporter determined by fluorescence spectrophotometry after excitation at 488 nm in the presence (dashed line) or absence (solid line) of reducing agent (TCEP).

Supporting Figure 8 Fig. 8.
The lysosomes and Golgi/TGN system are not involved in disulfide bond reduction of the folate-FRET reporter. (A) Neither the folate-FRET reporter nor its reduced fragments localize in lysosomes. KB cells were treated with LysoSensor Yellow/Blue DND-160 (1 mM) and the folate-FRET reporter (100 nM) for 45 min. After washing and further incubation for 12 h to allow reduction of the folate-FRET disulfide bond to reach completion, cells were imaged for BODIPY, rhodamine, and LysoSensor blue fluorescence by using the proper excitation and emission settings (488/520 nm, 545/600 nm, and 363/520 nm, respectively). (B) Effect of brefeldin A (BFA) on disulfide reduction of the folate-FRET conjugate. KB cells were first treated with BFA (2.5 ug/ml) for 1 h followed by 0.5 h of incubation with the folate-FRET (100 nM). After washing cells with fresh medium to remove unbound folate-FRET, BFA was added back and remained present throughout the entire observation period. Cells were incubated for another 12 h before imaging for BODIPY, FRET and rhodamine fluorescence (488/520 nm, 488/600 nm, and 545/600 nm, respectively). (C) Folate-FRET conjugate does not traffic to the Golgi/TGN system. KB cells were treated with a thioether (nonreducible) analog of the folate-FRET conjugate for 30 min. After washing and further incubation for 2 h, cells were stained with BODIPY FL C₅-ceramide complexed to BSA (5 ug/ml) according to the manufacturer’s instructions. Cells finally were imaged for BODIPY and FRET-induced fluorescence (488/520 nm and 488/600 nm, respectively). Virtually the same distribution pattern was seen by using a simpler folate conjugate, namely folate-rhodamine. Note the lack of coincident labeling in the overlay panel (Right). (Scale bar: 10 mm.)

Supporting Figure 9 Fig. 9.
Sorting of the folate-BODIPY fragment from released rhodamine following disulfide bond reduction. Cells were treated with the folate-FRET reporter for 0.5 h and the distributions of reduced folate-BODIPY fragment (A) and free rhodamine (B) were compared 12 h later by direct imaging of both dyes using excitations at 488 nm and 543 nm, respectively. (C) Overlay of A and B.

Supporting Figure 10 Fig. 10.
(Upper) Synthesis of the folate-FRET reporter. (Lower) ¹H-NMR spectrum (300 MHz) of the folate-FRET reporter. Three milligrams dissolved in 700 ml of D₂O, and the signal for DHO at 4.86 ppm was used as internal standard.

Supporting Text
Materials.
All reagents for the synthesis of the folate backbone were purchased from Novabiochem (San Diego, CA). Pteroic acid was a generous gift from Endocyte, Inc. BODIPY FL NHS ester, tetramethylrhodamine maleimide, LysoSensor Yellow/Blue DND 160, and transferrin Alexa 633 were purchased from Molecular Probes (Eugene, OR). Tetraethylrhodamine methanethiosulfonate was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was supplied by Pierce (Rockford, IL). DTNB, N-ethylmaleimide (NEM), brefeldin A, and colchicine were obtained from Sigma (St. Louis, MO). Folate-rhodamine was a generous gift from Erina Vlashi, Purdue University (West Lafayette, IN). Cells and Cell Culture.
KB cells, a human nasopharyngeal cell line, were grown continuously as a monolayer by using folate-free RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT) at 37˚C in a 5% CO₂/95% air-humidified atmosphere in the presence of ampicillin and streptomycin. Cells were grown in a glass-bottom 35-mm Petri dish (Mat Tek, Ashland, MA) for imaging studies. All cells were cultured to 50-60% confluence before each experiment. Specificity of all folate conjugates used in this study was established with confocal microscopy and flow cytometry by demonstrating that binding of the fluorescent folate conjugates was abolished upon addition of excess folic acid. Synthesis, Purification, and Analytical Characterization of Folate-FRET Reporter.
As shown in Fig. 10 Upper, Compound 1 was prepared by following standard Fmoc chemistry on an acid-sensitive trityl resin loaded with Fmoc-L-Cys (Trt)-OH, described in ref. 1 (adapted to the shown peptide sequence). The crude compound 1 was purified by HPLC using a VYDAC "protein and peptide C18" column, with 10 mM ammonium acetate (pH 5.0) and acetonitrile as the mobile phase.

HPLC-purified 1 (pH 2.5) was reacted in the presence of excess N,N-diisopropylethylamine (DIPEA) with tetraethylrhodamine methanethiosulfonate (Toronto Research Chemicals) in DMSO to afford compound 2. The reaction was instantaneous and quantitative as monitored by analytical HPLC (C18 reverse phase, mobile phase 1.0 mM sodium phosphate, pH 7.2, plus acetonitrile). The desired product was isolated by preparative HPLC as described above, except that the mobile phase was comprised of 1 mM sodium phosphate (pH 7.2).

The final conjugation was performed by mixing excess DIPEA with 2 (in DMSO) followed by addition of BODIPY FL NHS ester (predissolved in DMSO). After the reaction reached completion, compound 3 was isolated by preparative HPLC using the same condition as for compound 2, and its structure was confirmed by ¹H NMR in D₂O (see Fig. 10).

Synthesis, Purification, and Analytical Characterization of Folate-BODIPY.
Folate-BODIPY was prepared by reacting a cysteine-containing folate linker (2) with BODIPY FL maleimide (Molecular Probes, Eugene, OR). The desired product was isolated from the reaction mixture by using reverse phase HPLC with 10 mM ammonium acetate (pH 7.0) and acetonitrile as the mobile phase. The final product was characterized by ¹H NMR in D₂O. Note on Cell Line Used in These FR Trafficking Studies.
KB cells are a human cancer cell line reportedly derived from a nasopharyngeal carcinoma. Data on the rate of FR recycling in these cells in vitro suggest a recycling half time of ~6 h (3). Similar data on the rates of recycling of FR in other cell lines yield half times ranging from 0.5 h to 30 h (3, 4). How the kinetics of FR recycling affects the rate and location of disulfide reduction has not been explored.

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