NSC 185058, an inhibitor of ATG4B (a major cysteine protease), can also attenuate autophagic activity.

NSC185058 effectively suppressed starvation-induced autophagy in both cell types. GFP-LC3B labeled AVs were absent in starved MDA-MB468 cells when treated with NSC185058. The inhibitory effects of both NSC185058 on starvation-induced protein turnover were comparable to 3MA.

Mice were injected at 2 24 h intervals with vehicle or NSC185058, autophagy induced by 20 h fasting. Fasting resulted in an increase of GFP-LC3B AVs compared with ad libitum fed mice. NSC185058 significantly decreased the appearance of GFP-LC3B AVs compared with fasted mice.

Saos-2 transiently expressing FYVE-RFP was incubated in DMEM medium supplemented with amino acids and serum (Fed) or Krebs-Heinseleit medium lacking amino acids and serum (Starved). The starved cells were incubated in the presence and absence of the NSC185058 or NSC377071 for 4 h at which time the cells were examined by fluorescence microscopy. The numbers of red "dots" per cell was quantified using ImageJ software. Taking into account basal responses, those cells with 3 or more puncta were defined as PtdIns3K active.

Immunodeficient nu/nu nude female mice were injected subcutaneously with 6 × 10^6 Saos-2 (GFP-LC3B), shCon-Saos-2(GFP-LC3B) or shATG4B-Saos-2(GFP-LC3B) cells. Palpable Saos-2 (GFP-LC3B) tumors developed in 7 to 10 d, at which time, the mice were divided into 2 groups and injected IP on Monday, Wednesday, and Friday with either peanut oil vehicle or NSC185058 (100 mg/kg body weight) dissolved in peanut oil. Over time, the tumor dimensions were measured using calipers and volumes calculated using the formula: (W2 × L)/2.