Additional file 1 of Treatment of breast cancer with autophagy inhibitory microRNAs carried by AGO2-conjugated nanoparticles
Creators: name: Ozlem Unal
name: Akkoc, Yunus
name: Kocak, Muhammed
name: Nalbat, Esra
name: Dogan-Ekici, Asiye Isin
name: Acar, Havva Yagci
name: Gozuacik, Devrim
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cancer

description: Additional file 1: Figure S1. AGO2 protein binding improved MIR376B were abilized. (a) Overexpression of flag-AGO2 protein in HEK293T cells for 48 h and immunoprecipitation efficiency determined by pre- and flag-IP using flag antibody. p3Xflag was used as control transfection. (b) Comparative Taqman qPCR analysis of immunoprecipitated amount of MIR376B by RNA isolation from flag beads incubated with cell lysate of HEK293T cells with or without flag-AGO2 overexpression. (c) Determination of MIR376B level on flag-beads after 21 days of flag-AGO2 immunoprecipitation at room temperature by RNA isolation and Taqman qPCR. Taqman qPCR data was normalized using U6 small nuclear 1(RNU6-1) (U6) mRNA levels. Figure S2. Characterization of nanoparticles. (a) Zeta potential of SP-AH in PBS buffer (pH: 7.4) (b) Powder XRD pattern provided with the peak assignments of SP. (c) Thermogram of SP and PAA. (d) FTIR spectra of bare SPION, pure PAA and SP. Figure S3. (a) Measurement of AGO2 concentration of SP-A by Bradford assay. Standard curve generated from the absorbance of BSA at 595 nm as a function of BSA concentration (µg/mL). (b) Photoluminescence (PL) spectra of Dylight 650® at decreasing concentrations (2 × 10−2), 1 × 10−2, 4 × 10−2, 2 × 10−3, 1 × 10−3, 5 × 10−4 µM, λex: 655 nm, inset: Calibration curve of PL intensity vs concentration of DyLight 650®. PL spectra of (c) SP-AH (d) SP-F and (e) SP-AF. (λex: 655 nm, dilution factor: 20). Figure S4. Biocompatibility of SP and SP-AH nanoparticles in vitro and in vivo systems. (a) Determination of viability of 3 different breast cancer cells, MCF7, SKBR3 and MDA-MB-453, treated with increasing concentrations (5-500 µg/ml) of SP or SP-AH nanoparticles for 48 h by MTT cell viability assay. (b) Body weight change after 10 and 40 days in mice i.v. injected with SP, SP-AH (10 mg Fe per kg of mice) or equal volume of PBS. (c) Hematoxylin and eosin staining of mice tissues after 40 days of PBS, SP and SP-AH injections. Figure S5. Uptake and biodistribution of SP-AH nanoparticles in mice. (a) Iron amount in different mice tissues and tumor measured by Inductively Coupled Plasma (ICP) analysis one day after i.v. injection of PBS or SP-AH (10 mg Fe per kg of mice). (b) Ex vivo IVIS images of organs after one day of PBS or SP-AH i.v. injections. Figure S6. Determination of SP-AH nanoparticles on microRNA level in vitro. (a, b) QPCR analysis of SP-AH (150 µg/ml) treatment on MIR376B and its targets, BECN1 and ATG4C, after 48 h of treatment in SKBR3 (a) and MDA-MB-453 (b) cells. Control (CNT) cells were treated with equal amount of PBS with SP-AH treatment. QPCR data was normalized using U6 small nuclear 1 (RNU6-1) mRNA for MIR376B and GAPDH for ATG4C and BECN1 mRNAs. (mean ± SD of independent experiments, n = 3, *p