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. 2014 Nov 4;12(11):e1001988. doi: 10.1371/journal.pbio.1001988

Figure 8. Als acts downstream of GSK3β and upstream of SCF/Slimb/βTRCP E3 Ub ligase.

Figure 8

(A) als mRNA levels are downregulated upon als-dsRNA treatment. Stimulation of Wg signaling by inhibition of GSK3β (CHIR) or E1 ligase (PYR-41) causes an upregulation of als expression. (B and C) The positive Wg targets nkd and wf were induced upon GSK3β inhibition (bar 2 in [B and C]) and E1 ligase inhibition (bar 3 in [B and C]) in control cells. In als-dsRNA-treated cells, nkd and wf were strongly reduced in GSK3β-inhibited cells (bar 6 in [B and C]) and only mildly reduced in PYR-41-treated cells (bar 7 in [B and C]). Inhibition of the proteasome (MG-132) did not cause upregulated Wg target expression (bars 4 and 8). (D) Inhibition of GSK3β (CHIR) or E1 ligase (PYR) caused an increase of Arm protein levels in control cells (lanes 3 and 5); this increase was reduced upon als-dsRNA treatment (lanes 4 and 6); proteasome inhibition (MG132) caused elevated Arm levels in both control and als-dsRNA conditions (lanes 9 and 10). Combined depletion of ter94 and als could ameliorate Arm levels (lane 12, cf. lanes 13 and 14, which represent als depletion based on different als siRNA target regions). (E) Control expression levels are depicted as white bars. In wing imaginal discs (left bar diagram), als depletion caused a strong reduction of wf expression, a positive Wg target gene (lane 1), and an elevation of arr expression, a negative Wg target gene (lane 4). Combined als and ter94 depletion caused an elevation of wf (lane 2) or a decrease of arr (lane 5). No alteration was observed for the Hh target gene ptc (lanes 7–9). In Kc-167 cells (middle bar diagram), a co-depletion of als and ter94 caused an upregulation of the positive Wg targets nkd and wf (lanes 3 and 6), which were reduced upon als depletion (lanes 1 and 4). No alteration was observed for the Hh target gene ptc (lanes 7–9). Reduced nkd expression of als-depleted, GSK3β-stimulated Kc-167 cells (right bar diagram) could be ameliorated upon co-depletion of ter94. (F–I) The wing and eye phenotypes caused by the expression of the dominant-negative-like AlsHA were suppressed by the overexpression of Ter94HA. co-i, control siRNA.