Figure 1.
Stress granule inhibition by kinase inhibitors.
SH-SY5Y cells were treated with 1 mM paraquat overnight in the presence or absence of kinase inhibitors 1–25 (A) and 26–50 (B) from the Tocriscreen kinase inhibitor toolbox. The numbers of stress granules positive for TDP-43 and/or HuR were counted and compared to cultures treated with paraquat treatment alone. *P<0.05 compared to paraquat treatment alone. # on graph indicates ‘not done’.
Figure 2.
Stress granule inhibition by kinase inhibitors.
SH-SY5Y cells were treated with 1 mM paraquat overnight in the presence or absence of kinase inhibitors 51–80 (A) or selected inhibitors (B) from the Tocriscreen kinase inhibitor toolbox. The numbers of stress granules positive for TDP-43 and/or HuR (A) or TDP-43, HnRNP K, TIAR and/or HuR (B) were counted and compared to cultures treated with paraquat treatment alone. *P<0.05 compared to paraquat treatment alone. # on graph indicates ‘not done’.
Figure 3.
Effect of selected kinase inhibitors on TDP-43 expression.
SH-SY5Y cells were treated with paraquat overnight in the presence or absence of 10 µM LY294002 (#7, PI3K); olomoucine (#12, CDKs); ZM 449829 (#15, JAK3); GW 5074 (#17, Raf); SB 203580 (#19, p38); SB 415286 (#29, GSK3); arctigenin (#30, MEK); SB 239063 (#32, p38); (1 µM) aminopurvalanol A (#35, CDKs); TBB (#40, CK2); HA 1100 (#42, ROCK); BIBX 1382 (#43, EGFR); CGP 53353 (#44, PKC); arcyriaflavin A (#45, CDKs). Western blot analysis of TDP-43 expression was determined and represented as densitometric analysis of expression compared to untreated control. *P<0.05 compared to untreated control.
Figure 4.
Control of stress granule formation in retinoic acid-treated SH-SY5Y cells, and reversal of stress granule formation in non retinoic acid-treated cells by selected kinase inhibitors.
A: Stress granule inhibition by kinase inhibitors in retinoic acid-treated and non-treated SH-SY5Y cells. SH-SY5Y cells were treated with retinoic acid or left un-treated as described in Methods. Cells were then treated overnight with paraquat in the presence or absence of selected kinase inhibitors. The numbers of stress granules positive for TDP-43 were counted and compared to cultures with paraquat treatment alone. *P<0.05 compared to paraquat treatment alone. B: Ability of kinase inhibitors to reverse stress granule formation. Non-retinoic acid-treated SH-SY5Y cells were treated with paraquat overnight followed by 6 hr treatment in the presence or absence of selected kinase inhibitors. The numbers of stress granules positive for TDP-43 and/or HuR were counted and compared to cells treated with paraquat alone. *P<0.05 compared to paraquat treatment alone.
Figure 5.
Effect of selected kinase inhibitors on aggregation of CTF-TDP-43-GFP (219–414).
Non-retinoic acid-treated SH-SY5Y cells were transfected with CTF-TDP-43 (219-414)-GFP and incubated for 24 h. Selected kinase inhibitors were added for a further 24 h and the numbers of GFP-positive cytosolic inclusions determined and compared to untreated cells (no inhibitor) (A). The inhibitors examined were 10 µM LY294002 (#7, PI3K); olomoucine (#12, CDKs); GW 5074 (#17, Raf); SB 203580 (#19, p38); SP600125 (#23, JNK); (1 µM) SB 415286 (#29, GSK3); arctigenin (#30, MEK); (1 µM) SB 239063 (#32, p38); CGP 53353 (#44 PKC). *P<0.05 compared to untreated controls. B: Representative image of untreated and cells treated with olomoucine (#12, CDKs); SB 203580 (#19, p38); or SB 415286 (#29, GSK3). Green = TDP-43 (GFP), blue = DAPI, righthand column represents merged image of TDP-43 and DAPI. Arrows indicate cytosolic inclusions. Bar = 10 µm.