Figure 1.
Fluoresence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in live HeLa cells.
HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence (NanoParticles column) or the presence of the transfection reagents Matra, Lipofectamine 2000 or GeneJuice. Control cells were treated with neither nanoparticles nor transfection reagents. The same signal intensity range was used for all FITC images. The scale bar represents 50 microns.
Figure 2.
A statistical analysis of the level of FITC fluorescence in HeLa cells transfected with nanoparticles, in the presence of Lipofectamine, GeneJuice or Matra, compared to untransfected HeLa cells.
HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence or the presence of transfection reagents. The FITC fluorescence in the cells was quantified as described in the methodology, and the levels of fluorescence in nanoparticle-transfected cells compared to those in untransfected cells. This multi-level statistical analysis takes into account local variations within the cell population. The bars represent the region where there is a 95% probability that the mean fluorescence increase lies within it. The dot represents the mean calculated effect size. Bars not crossing the 1 line show significant evidence for an effect following transfection. The p-value indicates the probability that there was no difference in expression levels between the control and treatment samples. Hence, a lower p-value indicates a greater likelihood that there was a difference between the transfected and the untransfected cells.
Figure 3.
Confocal fluorescence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in fixed HeLa cells.
HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the presence of Lipofectamine. The overall FITC signal was reduced so that the level of fluorescence from the observed circular bodies was not saturating. The distribution of the LAMP1 protein extends through the majority of the cytoplasmic area thus provides an impression of the cell. In the overlayed image, the magenta regions indicate the presence of cellular bodies in which the FITC signal from the nanoparticles overlaps with the signal for the LC3A/B autophagosomal protein marker. The scale bar represents 25 microns and the images shown are all from a single z-plane (a 0.7 micron section).
Figure 4.
Reduction of hMTIIa gene expression in the presence of siRNA, ssRNA and ssDNA.
HeLa cells were untransfected (−), transfected with 5 nM control (C) or 5 nM hMTIIa (MT)-specific siRNA, ssRNA or ssDNA sequences using GeneJuice (Novagen). All samples were induced with 12.5 µM CdCl2. The level of hMTIIa gene expression (normalized to B2M) in untransfected and induced HeLa cells was defined as 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way.
Figure 5.
Levels of hMT-IIa in HeLa cells transfected with increasing concentrations of hMT-IIa or control ssDNA.
Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. All samples were induced samples with 12.5 µM CdCl2. Total hMT-IIa activity relative to B2M in cells transfected with varying levels of ssDNA was normalized to hMT-IIa-B2M expression in cells transfected with 0 nM ssDNA giving us a value for hMT-IIa activity in the presence of x nM ssDNA. The levels of hMT-IIa transcript in cells transfected with control ssDNA (C) was set to 1.0. The level of hMT-IIa gene expression (MT) in cells transfected with hMT-IIa ssDNA was compared to the levels of hMT-IIa in cells transfected with control ssDNA at each concentration providing a measure of the reduction in hMT-IIa activity in hMT-IIa ssDNA transfected compared to those transfected with control ssDNA. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. All samples were induced with 12.5 µM CdCl2. HeLa cells were transfected with hMT-IIa-specific ssDNA (ssDNA). Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in Table S2B.
Figure 6.
Levels of hMTIIa in HeLa cells transfected with gold nanoparticles.
Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. Samples were treated with 12.5 µM CdCl2. The level of hMTIIa gene expression (normalized to B2M) in untransfected and induced HeLa cells was defined as 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. Induced samples were treated with 12.5 µM CdCl2 (+Cd). HeLa cells were transfected with unfunctionalized (−), control (C) or hMT-IIa (MT) ssDNA-functionalized nanoparticles. Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in Table S2C.
Table 1.
Quantification of the levels of hMT-IIa transcript in HeLa cells after treatment with ssDNA-functionalized nanoparticles.