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. 2015 Apr 30;10(4):e0120348. doi: 10.1371/journal.pone.0120348

Fig 5. Loss of cathepsin L in Myc-induced pancreatic neuroendocrine tumors is associated with the elevated expression of markers of cell autophagy and apoptosis in vivo.

Fig 5

(A) Immunohistochemical analysis of LC 5 expression in the pancreata collected from the MycER TAM ;Bcl-x L and MycER TAM ;Bcl-x L ;CTSLKO animals untreated (Myc-OFF) or treated with TAM for the duration of 14 days (Myc-ON (14 days)). At least four animals were assayed for each genotype; immunohistochemical analyses done in duplicate; ten randomized fields per analysis were considered. The asterisks indicate areas of tumor tissue enlarged in the inset in the upper right corner. Note punctate LC3 staining in the MycER TAM ;Bcl-x L ;CTSLKO animals. The graph shows quantification of LC3 puncta per cells in described tumors (n = 4 (Myc; BclXL mice) and n = 3 (Myc, Bcl Xl, CTSL mcie) at least eight independent fields for animal were considered). Statistical analysis was done by unpaired Student’s t test. Scale bars, 50 μm. (B) Immunohistochemical analysis of intracellular localization of LC-3 and the lysosomal marker LAMP1 in pancreata collected from the MycER TAM ;Bcl-x L and MycER TAM ;Bcl-x L ;CTSLKO animals treated with TAM for the duration of 14 days determined by confocal microscopy (See Materials and Methods). White arrows marked the arrears of co-localization of LC3 and LAMP1 staining. At least three animals were assayed for each genotype; immunohistochemical analyses done in duplicate; nine randomized fields per analysis were considered. Graph shows LC3+ and LAMP1+ co-localization presented as the percentage of total LC3+ dots. *** p<0.0001 by the two-tailed Student’s t test. (C) Representative images from an IHC analysis of apoptosis assayed by activated caspase-3 (CC-3) staining in pancreata collected from the the MycER TAM ;Bcl-x L and MycER TAM ;Bcl-x L ;CTSLKO animals treated with control vehicle (Myc-OFF) or TAM for the duration of 1, 3 or 14 consecutive days. Islet area is outlined by dotted line. At least three animals were assayed for each genotype and time point. Graph shows quantification of CC3+ cells in islet area. Scale bars = 50 μm.