A) Co-culture gating strategy.

B) Retained CFSE in TC-1

Retained CFSE
3 **
→ **

(fold change)
**

CFSE MFI
2

→
→
→

→
→ 1

Events
CD45

SSC
SSC

IL-4
LPS+IFN-γ

IL-4→LPS+IFN-γ
IL-4→IFN-γ
TC-1
---

IL-4→PerHx+IFN-γ
IL-4→VLX+IFN-γ
FSC → CFSE → Zombie Violet → CFSE →

C) Macrophage long-term viability D) VLX impairs TC-1 proliferation E) VLX is washed

Macrophage Confluence TC-1 proliferation TC-1 proliferation

100
IL-4 IFN-γ±drugs 100 100
Control
TMZ
Confluence (%)

Confluence (%)

Confluence (%)
--- HX531 80
75 IL-4 75 CB839
IL-4→IFN-γ PerhX
VLX 60
IL-4→PerHx+IFN-γ
50 IL-4→VLX+IFN-γ
50 TC-1+s.n. wash 1
40
TC-1+s.n. wash 2
25 25
20 TC-1+s.n. wash 3
TC-1
0 0 0
0 20 40 60 80 100 0 10 20 30 40 50 60 70 0 8 16 24 32 40 48 56 64
Time (h) Time (h) Time (h)

F) Annexin V and PI staining in co-cultured TC-1 cells

--- LPS+IFN-γ IL-4 IL-4→LPS+IFN-γ IL-4→IFN-γ IL-4→PerHx+IFN-γ IL-4→VLX+IFN-γ
→
PI

Annexin V →
G) Apoptosis stage H) Annexin V and PI staining in TC-1 cells

Early Apoptotic Late Apoptotic Untreated Apoptotic cells

Staurosp.1μM (20h)
60 60 80
Annexin V PI (%)
Annexin V+ PI - (%)

*
Annexin V+ (%)

60
+

✱
40 40
+

40
→

20 20
20

0 0 0
PI

→
Untreated

STS (20h)
IL-4
LPS+IFN-γ

IL-4→LPS+IFN-γ
IL-4→IFN-γ
IL-4→PerHx+IFN-γ
IL-4→VLX+IFN-γ
---
IL-4
LPS+IFN-γ

IL-4→LPS+IFN-γ
IL-4→IFN-γ
IL-4→PerHx+IFN-γ
IL-4→VLX+IFN-γ
---

Annexin V