Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2016 Jan 15;30(2):220–232. doi: 10.1101/gad.270439.115

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2016 Akkari et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

PMC Copyright notice

Figure 4. — Macrophages expressing high levels of CtsZ infiltrate CtsB^−/−S^−/− tumors. (A) CtsZ is highly expressed in CtsB^−/−S^−/− RT2 tumors, and these tumors also show elevated macrophage numbers. Antibodies against CtsZ (red) and Iba1, a macrophage-specific marker (green), were used to identify cells expressing CtsZ and the presence of TAMs in wild-type RT2 and CtsB^−/−S^−/− RT2 tumors. CtsZ⁺ macrophages are particularly concentrated in the invasive edge of CtsB^−/−S^−/− RT2 tumors (evident in the lower images in the bottom panels). (B) Quantification of CtsZ⁺ cells in wild-type RT2 (n = 62) and CtsB^−/−S^−/− RT2 (n = 33) tumors relative to the total number of DAPI⁺ cells showed a significant increase in CtsB^−/−S^−/− RT2 tumors. (C) Quantification of Iba⁺ TAMs in wild-type RT2 (n = 108) and CtsB^−/−S^−/− RT2 (n = 28) tumors relative to the total number of DAPI⁺ cells showed a significant increase in TAMs in CtsB^−/−S^−/− RT2 tumors. (D) mRNA expression of the macrophage marker Cd68 was determined by qPCR analysis of wild-type RT2 (n = 4) and CtsB^−/−S^−/− RT2 (n = 6) whole tumors and revealed a significant increase in expression in CtsB^−/−S^−/− RT2 mice, consistent with the results in C. (E) Graph showing the macrophage content within and at the margin of individual tumors from wild-type RT2 and CtsB^−/−S^−/− RT2 mice, as determined by Iba1⁺ staining and according to each tumor invasive grade: encapsulated, IC1, or IC2 (as specified in Fig. 1E). (F) Low-magnification images of wild-type RT2 and CtsB^−/−S^−/− RT2 tumors stained with the macrophage-specific marker Iba1 (green) or hematoxylin and eosin (H&E), identifying the accumulation of TAMs at the margin of invasive CtsB^−/−S^−/− tumors compared with wild-type invasive tumors. (G,H) mRNA expression of the chemoattractants Csf-1 (G) and Ccl5 (H) was determined by qPCR analysis of wild-type RT2 (n = 4–5) and CtsB^−/−S^−/− RT2 (n = 5) whole tumors, which revealed a significant increase in expression of both genes in CtsB^−/−S^−/− RT2 mice. mRNA expression determined by qPCR was normalized to Ubiquitin C for each sample in D, G, and H. The graphs show mean ± SEM. Statistical significance was calculated by unpaired two-tailed Student's t-test. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Bars: A, 50 µm; F, 200 µm.