Immunohistochemistry of T47D xenograft tumors.

<p>Protein expression of A) Hemoxylin and Eosin staining under 40× magnification, B) gap junction proteins (Cx 43, 32, and 26) under 60× magnification, C) apoptotic proteins (caspase-3, -8, and -9) under 60× magnification and D) proliferative (Ki-67 and Cyclin D1) and survival proteins under 60× magnification in T47D xenograft tumors harvested after 7 IP injections treated with either DMSO (control), cisplatin, PQ1, PQ7, or a combination of cisplatin and PQs.</p

Immunohistochemistry of metabolic organs from nude mice.

<p>Protein expression of gap junction protein Cx43, the monokine induced by IFN-gamma (MIG), apoptotic protein (caspase 3) and survivin in A) kidney and B) liver from mice with T47D xenograft tumors harvested after 7 IP injections treated with either DMSO (control), cisplatin, PQ1, PQ7 or a combination of cisplatin and PQs (view image under 60× magnifcation).</p

Immunohistochemistry of organs harvested from nude mice.

<p>Protein expression of gap junction protein Cx43 in the uterus, heart, and brain (cerebral hemisphere and cerebellum) from mice with T47D xenograft tumors harvested after 7 IP injections treated with either DMSO (control), cisplatin, PQ1, PQ7 or a combination of cisplatin and PQs.(view image under 40× magnification).</p

Protein expression of T47D xenograft tumors.

<p>Protein expression of A) gap junction proteins (Cxs), B) apoptotic proteins (caspases), C) Cyclin D1 and survivin from T47D xenograft tumors harvested after 7 IP injections treated with either DMSO (control), cisplatin, PQ1, PQ7 or a combination of cisplatin and PQs. Actin and GADPH are loading controls. Numbers indicate the fold difference from control (top row) and cisplatin (bottom row). x = fold induction. <b>Bold</b> P-value is <0.05 compared to control or cisplatin (n = 3).</p

Cell isolation from hydrogel matrix.

<p><b>A.</b> Cells encapsulating peptide hydrogel diluted in MEM and cell pelleted with 5-minute centrifugation at 4°C. <b>B.</b> Storage G′ and loss G″ moduli hydrogel diluted 15 times at 1 Hz frequency and 1% shear strain at 4°C for 1 hour. <b>C.</b> Viable cells before cell encapsulation and after cell isolation. <b>D.</b> Cell viability before cell encapsulation and after cell isolation. *p<0.05, n = 3, error bars represent standard deviation.</p

Immunofluorescence assay of cells in 3D hydrogel.

<p><b>A.</b> MCF-7 cells were cultured in 2D monolayer for 2 days <b>(2D)</b> or in 3D hydrogels for 7 days <b>(3D)</b>. Immunofluorescence assay was performed. Red indicates actin and blue indicates DAPI-stained nuclei. <b>B.</b> MCF-7 cells were cultured in hydrogel for 5 days. After 5 days of colony formation, cells were cultured in 3D hydrogel for additional 2 days <b>(Control)</b> or treated with 30 µM cisplatin for 48 hours in hydrogel via “pre-mixed” method <b>(Treatment)</b>. Cells were isolated from hydrogels and immunostained with antibodies against actin (<b>a</b>), Ki67 (<b>b</b>), survivin (<b>c</b>), and cleaved caspase-3 (<b>d</b>). Red indicates protein of interest and blue indicates DAPI-stained nuclei. To obtain the 3D images, Z-stack images were taken (0.5–1 µm slices) and reconstituted in ZEN 2010 software. The results represent one of three independent experiments.</p

Western blot analysis of cells in 2D monolayer and 3D hydrogel.

<p>MCF-7 cells were cultured in 2D monolayer and 3D hydrogel for 7 days <b>(U)</b> or for 5 days and then treated with 30 µM cisplatin for 48 hours <b>(T)</b>. Cells were harvested and whole cell extracts were obtained. Western blot analysis of survivin, procaspase-3, and cleaved caspase-3 was performed. Actin was used as a loading control. The results represent one of three independent experiments.</p

Peptide hydrogelation in MEM.

<p><b>A.</b> Proposed mechanism of MEM-induced h9e peptide self-assembling hydrogelation (SEM image showing the nanofiber scaffold of the hydrogel matrix). <b>B.</b> Storage modulus G′ of 1, 2, and 3 mM peptide hydrogel during the hydrogelation at 37°C. <b>C.</b> SEM image of 1 mM peptide hydrogel. <b>D.</b> SEM image of 3 mM peptide hydrogel.</p

Dynamic rheological study of h9e hydrogel.

<p><b>A.</b> Storage modulus G′ of shear-thinning and recovery test of 1, 2, and 3 mM peptide hydrogel. <b>B.</b> Four times amplitude sweep test with shear strain from 1% to 500% and 1- 5-, and 10-minute breaks. <b>C.</b> Multiple times delivery of peptide hydrogel via pipette; hydrogel was shear thinning but reassembled quickly without permanently destroying hydrogel architecture. <b>D.</b> Temperature profile test of 1, 2, and 3 mM peptide hydrogel between 4°C and 50°C.</p

Culturing cells in h9e hydrogel.

<p><b>A.</b> LSCM image of cells distribution within hydrogel architecture. <b>B.</b> Morphology of cluster cell in 3D hydrogel matrix over 5 days. <b>C.</b> Morphology of cells growth on 2D plastic monolayer over 5 days.</p