Transfer System
Sensor verification
Imaging begins approximately 2 hours after the media is replaced with a fluorescent imaging medium. Get images every 1 hour. SW1990 cells were observed using 440 nm excitation light, and the cells turned from cyan to yellow.
Fig.A: AND logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The and logic gate pathways show that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is very low, and when both are present, the fluorescence value is high, indicating that both proteins are required to function in the pathway.
Fig.B: OR logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The or logic gate pathway shows that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is high, and when both are present, the fluorescence value is very low, indicating that two proteins are required to function in the pathway alone.
Fig C: NOR logic gate. The reporter was used at a concentration of 150 ng, and the fluorescence value was measured at 515 nm. The norm gate showed that the ordinate was the normalized fluorescence intensity value. When TEVP and TVMVP are present, the fluorescence value at 515 nm is very low, and when both are absent, the fluorescence value is high, indicating that it is necessary to have two proteins at the same time in the pathway to function.
ALKBH5
Scratch test to verify transfer ability
In the pancreatic cancer cell line SW1990, we modified ALKBH5 by siRNA knockdown and overexpression plasmids and performed a scratch test to detect the migration and crawling ability of pancreatic cancer cells.
A and D are blank control groups. B and E overexpress the ALKBH5 cell scratch assay; panels C and F are knockdown ALKBH5 cell scratch assays.
The results showed that the migration ability of the siALKBH5 (knockdown expression) group was stronger than that of the Ctrl group (blank control group), p<0.01. The migration ability of the ALKBH5-WT (overexpression) group was higher than that of the Ctrl group (blank control group).
Transwell experiment to verify invasion ability
In the pancreatic cancer cell line SW1990, we modified ALKBH5 by siRNA knockdown and overexpression plasmids and Transwell to detect the invasive ability of pancreatic cancer cells.
Ctrl is the blank control group, ALKBH5-WT is the ALKBH5 overexpression group, and siALKBH5 is the knockdown expression group.
The results showed that the invasive ability of ALKBH5-WT group was weaker than that of Ctrl group, p<0.01; the invasive ability of siALKBH5 group was stronger than that of Ctrl group, p>0.01.
WB experiment verification action pathway
Western blot analysis showed that the expression levels of β-catenin and p-GSK-3β in Wnt/β-catenin signaling pathway were up-regulated in the pancreatic cancer cell SW1990 compared with the control group (3xFlag-Vector). Significantly decreased, the total GSK-3β protein expression was unchanged, this result indicates that up-regulation of ALKBH5 can enhance the activity of GSK-3β and promote the phosphorylation of β-catenin, resulting in a decrease in intracellular β-catenin and inhibition of Wnt/ --catenin signaling pathway.
Delivery System
Exosomes (Fig. A), sw1990 (Fig. B), under confocal microscopy, it can be seen that the PKH26-labeled exosome showing red fluorescence is mainly located in the cytoplasm of sw1990, distributed in the perinuclear region, and most of the cells are visible. To the red fluorescent signal (Fig.C)
NTA analysis refers to nanoparticle tracking analysis. The experimental results showed that the number of particles of HEK293T cells overexpressing exosome booster at 100 nm and 250 nm was significantly higher than that of control cells (about 23 times that of control cells). This suggests that exosome potentiators can increase the number of exosomes secreted by 293T cells.
Infiltration System
Hypoxia induction system
We tested the induced expression of pHRE with CopGFP. CoCl(II) is used to produce anoxic conditions. The above figure shows the effect of pHRE on HEK 293T cells. The experimental results show that the fluorescence intensity of CopGFP increases with the increase of CoCl(II) concentration. At a concentration of 500 uM, the fluorescence intensity decreased significantly. This is the result of inhibition of cell viability by high concentrations of CoCl(II). A similar effect was observed in SW1990 cells. In addition, the fluorescence intensity of SW1990 is usually lower than that of HEK293T, which may be due to the poor expression efficiency of SW1990 as a differentiated cell.
Casp3 system
WB experiment verification mechanism
The picture shows the DNA electrophoresis photo. Apoptosis caused by activation of casp3, which is randomly cleaved between nucleosomes and degraded into 200 bp or an integral multiple thereof. Since the nuclear membrane is intact during DNA extraction, there is almost no DNA in the extract of normal cells, and the DNA in the apoptotic cell of apoptotic cells is extracted, and the DNA molecules of 200 bp in length or their integral multiples are electrophoresis. Strips.
Fluorescence microscopy for cell death
Different cells can produce different degrees of apoptosis under different conditions. Under the fluorescence microscope, the normal cells stained with Hochest33258 showed a uniform blue color, while the apoptotic cells showed dense dense stained particles with a cluster-like distribution.
Flow cytometry to detect the degree of apoptosis
Fig A: blank control, under high oxygen concentration, sw1990 cell status is normal cells
Fig B: blank control, under low oxygen concentration, sw1990 cell status is normal cells
Fig C: Negative control, at high oxygen concentration, after binding to empty exosomes, the sw1990 cell status is normal cells.
Fig D: Negative control, at low oxygen concentration, after binding to empty exosomes, the sw1990 cell status is normal cells.
Fig E: In the experimental group, after high oxygen concentration, after binding to the exosomes after transformation, the state of sw1990 cells was more early apoptosis.
Fig F: In the experimental group, after the combined oxygen concentration in the medium, the sw1990 cell status was more early apoptosis.
Fig G: In the experimental group, after low-oxygen concentration, after binding to the exosomes after transformation, the sw1990 cell status was more late apoptosis.
Basic Progress
Fig.A: AND logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The and logic gate pathways show that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is very low, and when both are present, the fluorescence value is high, indicating that both proteins are required to function in the pathway.
Fig.B: OR logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The or logic gate pathway shows that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is high, and when both are present, the fluorescence value is very low, indicating that two proteins are required to function in the pathway alone.
Fig C: NOR logic gate. The reporter was used at a concentration of 150 ng, and the fluorescence value was measured at 515 nm. The norm gate showed that the ordinate was the normalized fluorescence intensity value. When TEVP and TVMVP are present, the fluorescence value at 515 nm is very low, and when both are absent, the fluorescence value is high, indicating that it is necessary to have two proteins at the same time in the pathway to function.
