Supplementary MaterialsS1 Fig: MALDI MS spectra of Cyt c conjugated with Sulfo-LC SPDP. portions of the LC-SPDP crosslinker.(TIF) pone.0195542.s003.tif (85K) GUID:?D3690273-B788-41C3-A3A7-53E3391CBE40 S4 Fig: CD spectra of the unmodified and the crosslinked (S-LC-SPDP altered) Cyt c and Tf. Unmodified Cyt c and crosslinked Cyt c in secondary (A) and tertiary region (B). Unmodified Tf and crosslinked Tf in secondary (C) and tertiary region (D). (E) and (F) CD signals normalized and expressed as %.(TIF) pone.0195542.s004.tif (435K) GUID:?581BE900-D320-4C1A-A719-925298DFB523 S5 Fig: Cyt c-Tf conjugate dissociates when incubated under acidic or reducing condition. Dynamic light scattering of the Fluorouracil inhibitor Cyt c-Tf conjugate at the beginning of incubation (A) and after 12 Fluorouracil inhibitor h of incubation at room heat in 100 mM sodium acetate buffer (pH 5.5) (B) or 10 mM glutathione (C) Hydrodynamic radius of unmodified Cyt c (1.7nm) shown for comparison (D)After 12 h of incubation the conjugate dissociates into two peaks as seen under both acidic as well as reducing conditions.(TIF) pone.0195542.s005.TIF (276K) GUID:?AF8C0095-3F04-458D-B390-F73BD8E0E0B9 S6 Fig: Cyt c-Tf conjugate none reducible form (control) with Sulfo-SMCC crosslinker. DLS data showing the hydrodynamic radii of the conjugate before (A) and after (B) reduction with 10 mM TCEP.(TIF) pone.0195542.s006.tif (83K) GUID:?A3ED8EA9-2846-4AF2-906A-BFAF2A0CB8D3 S7 Fig: FPLC elution profiles of unreduced and reduced Cyt c-Tf conjugate from Superdex 200 increase column. (A) Elution peak of the unreduced Cyt c-Tf conjugate (B) Upon reduction with 10 mM TCEP, the Cyt C- Tf peak further resolves in to two peaks of constituents, Cyt C and Tf.(TIF) pone.0195542.s007.tif (101K) GUID:?874C7E76-2FA7-430B-93F0-CB62208BED9D S8 Fig: FITC labeled transferrin showing TfR in A549 cells and MRC5 cells. (A) Chromatogram showing the elution of FITC labeled protein from a Superdex 200 column. FITC absorbs at 495 nm while Tf absorbs at 280 nm. (B) Confocal image of FITC labeled transferrin internalized by A549 Fluorouracil inhibitor cells. The nucleus was stained with DAPI. (C) Z-stack image of A549 cells with FITC channel to differentiate the surface bound and internalized transferrin. Green punctate indicates the internalized FITC labeled transferrin. Fluorouracil inhibitor (D) Confocal image of MRC5 cells with FITC chanel after treatment with FITC labeled Tf. Compared to A549 a more diffused and weaker FITC transmission indicates a lower level of TfR in MRC5 cells.(TIF) pone.0195542.s008.tif (918K) GUID:?27981F3F-7013-40C2-A0D9-3F72DCC25D68 S1 Table: Peak area and retention volumes of the Cyt c-Tf purification. (DOCX) pone.0195542.s009.docx (20K) GUID:?D4F5E701-AB4B-4DA4-B078-C00DF2C1024A Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract One of the major drawbacks of many of the currently used malignancy drugs are off-target effects. Targeted delivery is usually one method to minimize such unwanted and detrimental events. To actively target lung malignancy cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is usually overexpressed by many rapidly dividing malignancy cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung malignancy cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that this conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only managed an Rabbit polyclonal to HDAC6 IC50 value similar to the well known drug cisplatin (50 M) in A549 malignancy cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression. Introduction Lung malignancy is one of the leading causes of death in the USA with a 5 12 months survival rate of only about 18% [1]. Non-small cell lung carcinoma (NSCLC) accounts for.