Caracterización mecánica de los hidrogeles de PEGDA-BD y PEGDA-Control

Lipofection was employed in these experiments using two separate cationic lipid reagents: Transfectin (Bio-Rad, Hercules, CA) and Lipofectamine RNAiMAX

(Invitrogen, Grand Island, NY). A full list of all commercially-obtained miRNA and siRNA molecules transfected in these experiments is included in Table 1. DNA constructs are described in section II below. During all transfections, antibiotics were omitted from cell culture media.

It is important to note that in all experiments where negative control RNA oligonucleotides (i.e miRNA mimics, miRNA inhibitors, target protectors) were transfected, universal negative controls were used. These commercially available universal controls have been designed to be minimally reactive with as few transcripts as possible based on complementarity analyses with mammalian transcriptome databases. These controls are not scrambled sequences and therefore do not necessarily have base composition identical to the experimental oligonucleotides for which they serve as controls.

In many experiments, changes in analytes levels were observed following transfection of negative control constructs as compared to mock transfection conditions. One possible explanation in these experiments is that changes in analyte levels

following negative control transfection represent global non-specific or stress-related expression changes secondary to high oligonucleotide burden or interruption of normal metabolic pathways. As such, comparing negative control to experimental condition is

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extremely appropriate in this case. An alternative explanation is that the negative control may have an inadvertent effect on analyte levels via sequence-specific

complementarity or indirectly by modulating expression of an upstream pathway. In this case, comparison of experimental condition to negative control is likely not appropriate. In general, statistical comparisons were made between the experimental treatment and either the negative control or the mock transfection depending on which control

condition exhibited the smallest difference in analyte levels compared to the experimental treatment. This approach was taken to avoid, as much as possible, inappropriate comparison between experimental treatments and negative control transfections.

In one set of experiments, DNA reporter constructs containing luciferase- expressing cassettes were transfected into HeLa, SK-N-SH and NT2N cells. For these experiments, HeLa cells (5x104 _{cells per well), SK-N-SH cells (1x10}5 _{cells per well) and} NT2N cells (1.6x104 _{cells per well) were cultured in white-walled 96 well plates, with} each well containing 100 µL of serum-supplemented media per well and transfected with 150 ng – 300 ng of reporter constructs using Transfectin. Transfection complexes were prepared by incubating DNA in a volume of serum-free media equivalent to 20 µL per well with 0.75 µL Transfectin per well for 15 – 20 minutes. Transfection complex- containing solution was then directly added to cells on-plate in serum-containing media. Luciferase assays were normally performed 48 hours after transfection.

In a separate set of experiments, HeLa cells were co-transfected with reporter constructs and miRIDIAN miRNA mimics (Dharmacon, Lafayette, CA). These co- transfections were performed by incubating HeLa cells cultured in 96-well plates (5x104 cells per well) with 150 ng reporter DNA and 40 nM miRNA mimic using 0.2 µL

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The remaining experiments were single transfections of Silencer Select siRNA (Applied Biosystems, Carlsbad, CA), miRNA mimics, miRNA locked nucleic acid (LNA) inhibitors (Exiqon, Woburn, MA), or miRNA Target Protectors (Qiagen, Valencia, CA) into HeLa or U373 cells using RNAiMAX reagent (primary human fetal neuron cultures discussed below). For most experiments, HeLa cells (1.35x105 _{cells per well) and U373} cells (7.5x104 _{cells per well) were cultured in 24-well plates and reverse transfected. In} reverse transfections, transfection complexes are added to cultures at the same time as cells are plated. So, in this transfection format, cells are initially transfected in

suspension until they settle and adhere to the plate. HeLa cells were generally transfected with 20 nM siRNA, 50 nM miRNA mimic, 100 nM – 1000 nM LNA miRNA inhibitor and 100-1000 nM miRNA Target Protector using 0.5 µL RNAiMAX per well. Transfection complexes were prepared in 50 µL Opti-MEM serum-free media

(Invitrogen, Grand Island, NY) with 10 – 15 minute incubation periods prior to mixing with cell suspensions. U373 cells were similarly transfected with 75 nM miRNA mimics using 3.5 µL RNAiMAX per well. In several cases, miRNA mimics were co-transfected into HeLa cells with siRNA or miRNA target protectors. In these cases, RNAiMAX levels were boosted to 1µL per well to account for the increase in nucleic acid content.

Multiple batches of human fetal brain cultures were transfected at DIV 17 in 24- well plates. Cultures were transfected with 20 nM siRNA, 150 nM miRNA mimics, and 1000 nM LNA miRNA inhibitors using 1.25 µL RNAiMAX per well. bFGF was omitted from media during transfections. In one series of experiments, human fetal brain

cultures were transfected with miRNA mimics in the presence of 150 µM deferroxamine mesylate (DFO) (Sigma-Aldrich, St Louis, MO). The appropriate volume of DFO was prepared from a 5 mg/mL stock solution in PBS and added to cell culture plates approximately one hour prior to transfection.

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In all experiments employing transfection of small RNA oligonucleotides,

transfection efficiency was assessed qualitatively by including a siRNA transfection (20 nM) against the gene product of interest. These siRNA were validated in HeLa cells as capable of reducing APP or BACE1 protein and mRNA expression to less than 5% of mock or negative control siRNA transfections.

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Table 1: List of transfected siRNA and miRNA molecules

siRNA Reagents

Targeted mRNA Vendor siRNA ID APP Applied Biosystems s1500 BACE1 Applied Biosystems s24219 Dicer Applied Biosystems Custom AGO2 Applied Biosystems Custom IRP1 Applied Biosystems s672 Negative Control #1 Applied Biosystems 4390843 miRNA Mimics

miRNA Name Vendor Catalog Number

hsa-miR-1 Dharmacon C-300585-05 hsa-miR-101 Dharmacon C-300518-07 hsa-miR-153 Dharmacon C-300615-07 hsa-miR-346 Dharmacon C-300712-03 hsa-miR-339-5p Dharmacon C-300707-07 hsa-miR-29b Dharmacon C-300520-05 Negative Control Mimic #1 Dharmacon CN-001000-01 Negative Control Mimic #2 Dharmacon CN-002000-01 miRNA Antisense LNA Inhibitors

Targeted miRNA Vendor Catalog Number

miR-101 Exiqon 412942-08

miR-153 Exiqon 410076-08

miR-346 Exiqon 411373-00

Negative Control A Exiqon 199004-00 Custom miRNA Target Protectors

miRNA Target Site Vendor

miR-346 APP Target Site Qiagen miR-339-5p BACE1 Target Site 1 Qiagen miR-339-5p BACE1 Target Site 2 Qiagen Negative Control Target Protector Qiagen

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D. Harvest and preparation of cell culture lysate for RNA and protein analyses