Figure 1.
Fluorescence signal per lysate after transduction.
The fluorescence intensity of the lysed cells per well was measured in a multiwell fluorescence reader. The intensities were normalized to the mean intensity produced by the transfection using Proteoducin as transduction reagent. The sarcoma cells were transduced with Atto488-BSA using different transduction reagents. Untreated sarcoma cells were measured as controls. As a control for standard endosomal uptake, sarcoma cells were treated with DMEM containing 0.5 µg Atto488-BSA without a transduction reagent (termed as Atto488-BSA). Error bars represent the 95% confidence intervals of the means. Each transduction reagent was tested in at least 6 independent experiments.
Figure 2.
Fluorescence signal per cell after transduction.
Sarcoma cells were transduced with Atto488-BSA using different transduction reagents. The fluorescence was measured in a flow cytometer, untreated cells were used as control and standard endocytosis was measured by treating the cells with 2 µg Atto488-BSA containing DMEM. Error bars represent the 95% confidence intervals of the means depicted. Measurements were set to 20000 gated events.
Figure 3.
Effects of PCI of transduced Atto488-BSA in sarcoma cells.
(A) Phase contrast picture of living sarcoma cells after protein transduction using the transduction reagents Chariot, Proteoducin or Endo-Porter. (B) Fluorescence signals inside of the same sarcoma cells. The signals are predominantly punctate with a perinuclear localisation. Only in Endo-Porter transduced cells a cytosolic signal is visible. (C) Fluorescence signals of the same cells after 200 seconds blue light exposure. The Atto488 signal has spread throughout the cytosol. In cells transduced via Chariot and Proteoducin, a clear fluorescence distribution change is visible. The existing cytosolic signal in Endo-Porter transduced cells is increased although the effect is weaker compared to the other reagents.
Figure 4.
Cytotoxicity of the PCI treatment.
To measure cytotoxicity of the PCI application sarcoma cells were stained with trypan-blue 3 h, 24 h and 48 h after various treatments. Cells exposed to light (L), TPPS4 (PS) or with both show no striking changes in the percentage of trypanblue positive cells compared to untreated controls. Cells transduced with Proteoducin (T) show an initial increase of dead cells. Additional treatments with the TPPS4 photosensitizer or complete PCI did not change the percentage of trypanblue positive cells. Each experiment was performed 3 times (n = 300−900). The error bars represent the 95% confidence intervals of the means.
Figure 5.
Change of signal distribution after PCI treatment.
The median of the pixel gray values was calculated in fluorescence images of Atto488-BSA transduced sarcoma cells using different transduction reagents. Single cells were defined as region of interest in the ImageJ software and the median of the gray values was compared between cells before and after PCI treatment as described in Methods. The means of the medians of the gray values before and after PCI treatment are given. Error bars represent the 95% confidence intervals (n = 10). CPP-based transduction reagents (Proteoducin and Chariot) show high signal distribution changes. Reagents based on lipids (Proteofectene, Lipodin-Pro, ProJect and Pro-DeliverIN) also show changes but with partially lower significance. Reagents described as non-lipid or cationic polymers (TransPassP and TurboFect) did not lead to a signal spreading as a reason of a PCI treatment. The cytosolic signals produced by transduction with the endosomolytic reagent Endo-Porter was increased significantly by further disruption of endo- and lysosomes by PCI treatment.