Figure legends
Fig. 1. IL-28B therapy efficiently suppressed subcutaneous MC38
tumors. C57BL/6 mice (6-8 weeks) were inoculated subcutaneously with
1×106 MC38 tumor cells. From day 0, tumor volume was
measured every 48 h, and IL-28B (2.5 µg/mouse) was injected into the
tissue surrounding the tumor once a day. All mice were assayed on day
18. (A) The schedule of IL-28B in the treatment of mouse colon tumors.
(B) Representative images of each group of tumors after IL-28B
treatment. (C) Based on tumor volume, the IL-28B effectively inhibited
the tumor growth compared with the control group. (D) The weight of each
group of tumors is shown. (E) H&E staining of paraffin-embedded tumor
sections. IL-28B protein increased tumor tissue necrosis, ×200 original
magnification; n = 5; **P < 0.01; *** P < 0.001.
Fig. 2. IL-28B reduces M2
macrophages in tumor tissues. (A, B) Representative
FACS plots and ratio of macrophages
(CD11b+F4/80+CD206+)
in tumors (CD45 events). (C-I) Changes in M1 macrophages
(CD11b+F4/80+CD86+),
MDSC (CD11b+Gr-1+), CD4 cells
(CD3+CD4+) and CD8 cells
(CD3+CD8+) in tumors. (J)
Immunofluorescence staining of infiltrated F4/80+CD206+ M2 macrophages in tumors. Red, anti-F4/80 Ab;
green, anti-CD206 Ab; yellow, F4/80 and CD206 merged; blue, DAPI.
Original magnification: ×200. **P
< 0.01; n.s. denotes, P > 0.05.
Fig. 3. Effect of IL-28B on
M1 and M2 macrophage polarization in vitro . (A) IL-28B treatment
of M2 macrophages and experimental workflow. Briefly, bone marrow cells
were isolated from the femur and tibia of mice in PBS. Then, cells were
cultured in complete DMEM containing GM-CSF (10 ng/mL), and IL-28B
protein (0, 100, 200, and 400 ng/mL) in treatment groups. After four
days, media was supplemented. On the seventh day, BMDMs were harvested,
IL-4 (10 ng/mL) and IL-13 (10 ng/mL) were used to polarize BMDMs to M2
macrophages for subsequent experiments. M1 and M2 macrophages were
similarly treated with IL-28. (B - E) The proportions of M1 and M2 in
BMDMs were determined using flow cytometry. Data are expressed as mean ±
SEM.*P < 0.05, **P < 0.01 and ***P <
0.001.
Fig. 4. IL-28B treatment
inhibits the expression of marker cytokines in M2 macrophages. (A)
Enzyme-linked immunosorbent assay (ELISA) was used to detect the
expression of cytokines, TGF-β (supernatant), and Arg-1 (intracellular).
The cell culture supernatants and total cellular proteins were collected
after 24 h of polarization of M2 macrophages. (B) Real-time PCR was used
to detect cytokine mRNA expression in M2 macrophages. The total cellular
RNA was collected after 4 h of polarization of M2 macrophages. Data are
expressed as mean ± SEM. *P < 0.05; **P < 0.01 and
***P < 0.001; n.s.
denotes, P > 0.05.
Fig. 5. IL-28B protein downregulates the STAT3 and JNK
signaling pathways in M2 macrophages. The total cell protein was
collected after 1 h of polarization of M2 macrophages. The levels of
STAT3, p-STAT3, p-JNK, and JNK were analyzed by western blot analysis.