Fig 1.
Overexpression of TDP-43 on p65 activation.
A. p65 (Green) is translocated into the cells’ nuclei (Blue) after 30 minutes of 10ng/ml TNF-α treatment (n = 3 repeats). B. MCF-7 cells were transfected with various doses (indicated on the right) of wild type TDP-43 tagged with mCherry expressing plasmids (middle panel, red) and were treated with 10ng/ml TNF-α for 30 minutes. In the top right corner, insets of higher magnification show the localization of p65 and nuclear TDP-43 staining (n≥3 independent experiments). C. The quantification of B. D. MCF-7 Cells were transfected with 4μg plasmid encoding TDP-43 tagged with mCherry or mCherry empty plasmid (control) and treated with 10ng/ml TNF-α for 30 minutes. Nuclear proteins were isolated and detected by western immunoblotting. Histone H3 was used as a nuclear marker. The relative density was the average of 3 individual experiments. E. MCF-7, Neuro 2a and BV2 cells were transfected with Wild type TDP-43 tagged with mCherry expressing plasmids (middle panel, red). MCF-7 and Neuro 2a cells were treated with 10ng/ml TNF-α for 30 minutes; BV2 cells were treated with 10μg/ml LPS for 30 minutes. Arrows indicate the blocked p65 nuclear translocation by Wt TDP-43 and stars indicate the normal p65 nuclear translocation with Wt TDP-43 overexpression. n≥3 independent experiments. F. The plasmid encoding TDP-43 tagged with mCherry or mCherry empty plasmid (control) was cotransfected along with NF-κB-luc (containing the wild type NF-κB-binding site). Cells were treated with 10ng/ml TNF-α for 30 minutes. Luciferase activity was measured after 24 hours. The plotted error bars represent mean±SEM from 3 independent experiments; *p < 0.05 compared to control, one-way ANOVA. ** p < 0.05 compared to the TNF-α treated control samples, Student's t test.
Fig 2.
Knockdown of endogenous TDP-43 increases nuclear p65 protein levels after TNF-α treatment.
A. MCF-7 cells were transfected with TDP-43 siRNA or scrambled siRNA, treated with 10ng/ml TNF-α for 30 minutes. Nuclear proteins were analyzed by western blot and detected with antibodies against TDP-43, p65 and Lumin B1 as indicated. Lumin B1 was used as nuclear marker. n = 3 independent experiments. B. The relative density of p65 as shown in A. n.s not significant, *p < 0.05 compared to control, one-way ANOVA, ** p < 0.05.
Fig 3.
Overexpression of TDP-43 accelerates IκB degradation.
Cells were transfected with wild type TDP-43 expressing plasmids (tagged with mCherry) or mCherry empty plasmid (control) and were treated with 10ng/ml TNF-α for the indicated periods. Total proteins were extracted for Western blot analysis using antibodies as indicated. n = 3 independent cultures; A. 30 min after TNF-α treatment, the degradation of IκB occurred in both control group and TDP-43 overexpression group. B. Overexpression of TDP-43 facilitates the degradation of IκB within 5 minutes after TNF-α treatment.
Fig 4.
Overexpressing TDP-43 inhibits NF-κB activity by competing for the nuclear transporter.
A. Cells were transfected with wild type TDP-43 expressing plasmids (tagged with mCherry) or mCherry empty plasmid (control) and were then treated with 10ng/ml TNF-α for 30 minutes. Cell lysates were subjected to coimmunoprecipitation and western blot for the presence of p65 and TDP-43. 10% cell lysate were reserved as input. p65, TDP-43 and importin α3 (KPNA4) were used to detect input. n = 3 repeats. B. The relative density of p65 as shown in A (IP), *p<0.05. C. The relative density of wild type TDP-43 as shown in A (IP), *p<0.05.
Fig 5.
The blockade of NF-κB nuclear translocation by TDP-43 can be prevented by overexpression of p65.
MCF-7 cells were transfected with wild type TDP-43 expressing plasmids (top row) alone or cotransfected with TDP-43 and p65 expressing plasmids (bottom row). Then MCF-7 cells were treated with TNF-α for 30 minutes. The p65 was found co-localized with TDP-43 in the nuclei (bottom row). n = 3 independent experiments.
Fig 6.
The NLS mutated TDP-43 does not affect p65 nuclear translocation.
MCF-7 cells were transfected with TDP-43 ΔNLS expressing plasmids (middle panel, Red). After 30 minutes of TNF-α treatment, labeled p65 (Left panel, Green) was found in the nuclei. n = 3 independent experiments.
Fig 7.
Schematic hypothetical modes for TDP-43 and p65 competition.
The presence of TDP-43 in the cytosol may compete with p65 for importin α3 (cytoplasmic mode) and therefore to inhibit the nuclear localization of p65. An alternative mode (nuclear mode) shows that nuclear TDP-43 may occupy importin α3 to prevent its exit and therefore to reduce the availability of free importin α3 in the cytosol for p65 nuclear translocation.
Fig 8.
The competition of importin α3 (KPNA4) occurs in both the cytoplasm and nucleus.
The nuclear and cytoplasmic proteins of MCF-7 cells were separated after overexpression of wild type TDP-43 and TNF-α stimulation. The top panel shows the protein levels of total TDP-43 in each fraction. N: nucleus. C: cytoplasm. The bottom shows that TDP-43 interacted with importin α3 (KPNA4). p < 0.05, Student's t test.