Liposomal delivery of siRNA efficiently downregulates FTH1 expression in GICs.

(A) Western blot analysis of FTH1 expression for T3691 cells transfected with siRNA: liposome ratio of 2μg:4μl. (B) Densitometric quantification of T3691 cells in (A) showed significant reduction in FTH1 expression relative to control siRNA treated cells at 24h (57%,p<0.01), 48h (86%, p<0.001) and 72h (79%,p<0.001).(C) Western blot analysis of FTH1 expression in T387 cells transfected at ratios of 2μg:4μl. (D) Densitometric analysis of T387 cells in (C) showed comparatively poor knockdown at 24h (9%, p<0.05) and 48h (49%, p<0.001) with the maximum knockdown achieved at 72h post transfection (63%, p<0.001). (E) Western blot of FTH1 expression in T387 cells transfected at 2μg:8μl. (F) Densitometric analysis of (E) showed that changing siRNA: liposomes ratio to 2:8 in T387 cells improved knockdown significantly at 24h (45%, p<0.001), 48h (66%, p<0.001) and 72h (44%, p<0.01). Expression levels were normalized to endogenous actin levels and then to expression of FTH1 in liposome treated controls. Percentage values are relative to control siRNA from three independent experiments.</p

Loss of FTH1 impairs DNA repair and colony formation ability of GICs following radiation.

(A) Total phospho-ϒH2AX levels were evaluated by western blot at 48h post-transfection with FTH1 siRNA. (B) Quantification of phospho-ϒH2AX levels in T3691 and T387 cells showed a 3 fold increase in phosphorylation of ϒH2AX relative to control siRNA treated cells. (C) phospho-ϒH2AX foci formation in T3691 was assessed 24h post transfection (1hr, 0Gy), 1hr and 24h post radiation at 2Gy. Compared to control siRNA, T3691 GICs showed a 2.3 fold increase in number of foci/nuclei (***p<0.001) in the absence of radiation and further continued to retain foci starting at 1hr (2 fold, ***p<0.001) and upto 24h post radiation (1.6 fold, **p<0.01). (D) T387 cells treated similarly, also showed a 1.9 fold increase in foci number (***p<0.001) at 0Gy, but showed no significant change relative to controls siRNA treated cells upon radiation. (E) T3691 GICs failed to form colonies and showed significant reduction in colony formation compared to control siRNA treated GICs (100%, ***p<0.001) in the absence of radiation. (F) T387 GICs showed a comparatively moderate decrease in colony formation without radiation at 0gy (32%, p<*0.05). Colony forming ability of FTH1 siRNA silenced GICs was further impaired by radiation (52%,*p<0.05) compared to control siRNA and compared to its un-irradiated counterpart (38%, #p<0.05). Data from three experiments was normalized to liposome treated controls.</p

FTH1 knockdown lowers cell viability of T3691 but not T387 GICs following radiation.

GICs were transfected with FTH1 or control siRNA at siRNA (μg): liposome (μl) ratios of 2:4 (T3691) or 2:8 (T387) for 24h, followed by no radiation (0Gy) or radiation at 4Gy and 8Gy. Cell viability was assessed 24 and 48 hours after radiation exposure using MTS assay. (A, B) In T3691 GICs, at both 24h (a) and 48h (b) post radiation, cell viability in FTH1 siRNA treated group was significantly reduced relative to control siRNA irrespective of radiation at 0Gy, 4Gy and 8Gy (*** = p<0.001). FTH1 siRNA treated T3691 cells radiated at 8Gy showed an additive decrease in cell viability relative to un-irradiated controls (0Gy) (Λ = p<0.01) as well as cells radiated at 4Gy (Λ # = p<0.05). However, there was no significant reduction in viability at 4Gy relative to un-irradiated controls (0Gy). (C) 24h post radiation, T387 GICs also showed significant decrease in cell viability at all three doses (*** = p<0.001). (D) T387 GICs showed no decrease in cell viability at 48h post radiation (ns = not significant). T387 GICs showed no significant change in cell viability after radiation exposure relative to un-irradiated controls (ns = not significant). Data from three experiments was normalized to liposome treated controls.</p

Liposome characterization.

(A) Mean (±SD) particle size, polydispersity index and (B) zeta potential of three batches of liposomes was 94.8 ±16.4 nm in diameter (d.nm) and 43.56±5.06 millivolt (mV) respectively. (Table 1) Zeta potential of siRNA (μg): liposome (μl) complexes used for transfection of T3691 (2μg:4μl) and T387 (2μg:8μl) GICs was determined to be anionic and cationic respectively. (C) DiI labeled liposomes (magenta) transfected show colocalization in EEA1 labeled early endosomes (yellow) indicating endosomal uptake mechanism. (D) Transfection with DiI-liposome (magenta): siGLO (yellow) complexes showed efficient uptake and localization in cytosol indicating endosomal escape. Nuclei were counterstained with DAPI (cyan). Arrowheads point to areas of EEA1-DiI colocalization (C) or siGLO foci (D). Scale bar = 10μm.</p

FTH1 knockdown is detrimental to GIC survival.

(A) Knockdown of FTH1 led to increased extracellular LDH release in T3691 (268%, p<0.001) and T387 (57%, p<0.01). (B) Executioner caspase 3/7 activity was similarly elevated in T3691 (62%, p<0.01) and T387 cells (63%, p<0.01). (C) Mitochondrial damage was assessed by TOMM20 staining (yellow) at 24h post-transfection with FTH1 siRNA showing perinuclear localization (yellow). Scale bar = 10μm. (D) Decrease in mitochondrial mass, measured by quantifying TOMM20, occurred in both T3691 (72%, p<0.001) and T387 (70%, p<0.001) GICs. (E) Staining for stemness marker Nestin (yellow) with nuclei were counterstained with DAPI (cyan). Scale bar = 10μm. (F) Nestin positive cell were significantly reduced in T3691 GICs at 48h (71.2%, p<0.001) but not in (F) T387 GICs. Number of Nestin positive cells was normalized to untreated controls. Data from three experiments were normalized to liposome treated controls (a, b, d). Percent values are relative to control siRNA.</p

Zeta potential analysis of siRNA: liposome complexes used for GIC transfection.

Zeta potential analysis of siRNA: liposome complexes used for GIC transfection.</p

Versican increases fibroblast-mediated contraction of a collagen lattice.

<p>(A) Representative images of contracted collagen gels are shown along with the quantified surface areas of contracted gels. Versican significantly increased the fibroblast-mediated contraction of collagen gels (14.9 ± 0.7% vs 24.8 ± 1.8% of initial gel area, p<0.05). (B) Confocal imaging demonstrated the versican-transfected fibroblasts to be elongated, interconnected, and to have increased stress fibre formation in collagen gels (arrows, red in overlay). Versican was found to localize to the pericellular matrix surrounding elongated cells (arrowheads, green in overlay). (C) A Z-stack image reveals versican (green) forms a pericellular coat around cell protrusions in versican-transfected fibroblasts, suggesting it may be well-situated to influence biological events at the cell membrane. (Scale bars = 23.00 μm in B, 12.00 μm in C, * denotes p<0.05)</p

Versican increases N-cadherin expression.

<p>(A) Light microscopy shows no major change in cell morphology in versican-transfected fibroblasts at both sub-confluent and confluent densities. (B) Representative Western blot showing increased expression of N-cadherin in versican-transfected cells. (C) Confocal microscopy confirmed the increased N-cadherin expression in versican-transfected cells. (Scale bar = 12.00 μm).</p

Versican V1 overexpression induces a myofibroblast-like phenotype in cultured fibroblasts

Background: Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts. Methods and Results: The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle a-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2. Conclusions: Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts

Versican increases smooth muscle α-actin expression.

<p>(A) Versican induced a 1.38 ± 0.15 fold increase in smooth muscle α-actin mRNA expression, p<0.05. (B) Versican induced a 1.47 ± 0.10 fold increase in smooth muscle α-actin protein expression, p<0.05. (C) Immunofluorescent staining confirmed smooth muscle α-actin expression was increased in the versican-transfected cells. (D) Versican induced a 2.93 ± 0.22 fold increase in collagen III mRNA expression, p<0.05. (Scale bar = 47.00 μm, * denotes p<0.05)</p