FIGURE 5:

Atg16L1 regulates hormone secretion in PC12 cells independently of their autophagic activity. (A) Effect of Atg16L1 knockdown on hormone secretion from PC12 cells. NPY-T7-GST secretion assays were performed as described in Materials and Methods. The results are expressed as percentages of high-KCl-dependent NPY secretion from control PC12 cells. Bars indicate the means and SE of three determinations. Note that Atg16L1 knockdown resulted in a dramatic reduction in both low-KCl- and high-KCl-dependent NPY secretion (shaded bars) in comparison with the control PC12 cells (closed bars). **p < 0.01 (Student's unpaired t test). Under our experimental conditions, knockdown of Atg16L1 had little effect on the total amount of hormone (NPY-T7-GST; inset), although the total amount of NPY-T7-GST in Atg16L1 shRNA #2–expressing cells was somewhat decreased. Total cell lysates of PC12 cells that had been cotransfected with pShooter-NPY-T7-GST and pSilencer-Atg16L1 (or control empty pSilencer vector) were subjected to 10% SDS–PAGE followed by immunoblotting with anti-T7 tag antibody and anti-actin antibody. The knockdown efficacy of Atg16L1 shRNAs is shown in Figure 1C. (B) Effect of Atg13 knockdown or ULK1 knockdown on hormone secretion from PC12 cells. NPY-T7-GST secretion assays were performed as described. N.S., not significant in comparison with the control cells. Note that neither Atg13 knockdown nor ULK1 knockdown affected hormone secretion but that knockdown of each of them significantly inhibited starvation-induced autophagy (Supplemental Figure S5A). Total cell lysates of PC12 cells that had been cotransfected with pShooter-NPY-T7-GST and pSilencer-Atg13 (pSilener-ULK1 or control empty pSilencer vector) were subjected to 10% SDS–PAGE followed by immunoblotting with anti-T7 tag antibody and anti-actin antibody (inset). Under our experimental conditions, inhibition of autophagy had little effect on the total amount of NPY-T7-GST. (C) Effect of Rab33A knockdown on hormone secretion from PC12 cells. Rab33A shRNA was transiently coexpressed with NPY-T7-GST in PC12 cells. The NPY-T7-GST secretion assays were performed as described in A. Note that Rab33A knockdown caused inhibition of both the low-KCl-dependent and high-KCl-dependent NPY secretion (shaded bars). **p < 0.01 (Student's unpaired t test). Total cell lysates of PC12 cells that had been cotransfected with pShooter-NPY-T7-GST and pSilencer-Rab33A (or control empty pSilencer vector) were subjected to 10% SDS–PAGE followed by immunoblotting with anti-T7 tag antibody and anti-actin antibody. Under our experimental conditions, knockdown of Rab33A had little effect on the total amount of hormone (NPY-T7-GST; inset), although the total amount of NPY-T7-GST in Rab33A shRNA #2–expressing cells was somewhat decreased. The results shown are representative of at least two independent experiments.