TABLE 1.
ICOS‐induced ICOS‐L downmodulation: Effect of inhibitors of endocytosis, cell cytoskeleton, protein kinase C, lysosomal function, autophagy, Src, MAPK, FAK, and PI3 kinases, or Calcium chelators
| Endocytosis | Inhibitor | Inhibits ICOS‐L Downmodulation a , b |
|---|---|---|
| Clathrin‐Mediated Endocytosis | Chlorpromazine (50 µM) | +/‐ |
| Caveolin‐Mediated Endocytosis | Genistein (100 µg/mL) | + |
| Dynamin GTPases/Endocytosis | Dynasore (100 µM) | ++ |
| Cholesterol Depletion/Lipid Raft‐Dependent Endocytosis | Methyl‐β‐Cyclodextrin (10 mM) | ++ |
| Cytoskeleton | Inhibitor | Inhibits ICOS‐L Downmodulation a , b |
|---|---|---|
| Actin polymerization | Cytochalasin D (10 µM) | ++ |
| Myosin ATPase | Blebbistatin (30 µM) | – |
| Myosin Light Chain Kinase | ML‐7 (10 µM) | – |
| Tubulin (microtubule growth) | Colchicine (20 µM) | – |
| ROCK (Rho‐dependent kinase) | H‐1152 (5 µM) | – |
| Protein Kinase C | Inhibitor | Inhibits ICOS‐L Downmodulation a |
|---|---|---|
| Protein Kinase C α, β, γ, δ, ε | Gö6850 (Bisindolylmaleimide I) (1‐10 µM) | ++b |
| Protein Kinase C α, β, γ, δ, ε | Ro‐31‐8220 (Bisindolylmaleimide IX) (5 µM) | ++ c |
| Metabolic and Divalent ion blockers | Inhibitor | Inhibits ICOS‐L Downmodulation a , b |
|---|---|---|
| Low Temp (4°C) | ‐ | ++++ |
| Respiratory Chain | NaN3 (15 mM) | – |
| Ca++, Mg++ Chelation | EDTA (10 mM) | – |
| Ca++ Chelation | EGTA (10 µM) | – |
| Lysosomal function, Autophagy, Calcium | Inhibitor | Inhibits ICOS‐L Downmodulation a |
|---|---|---|
| Lysosomal proton pump/Autophagosome‐lysosome fusion | Bafilomycin A (0.5 µM) | – |
| Lysosomal/Autophagy inhibitor | Chloroquine (100 µM) | – |
| Golgi Transport | Brefeldin A (40 µM) | – |
| Transglutaminase 2 | Cystamine (500 µM) | – |
| Calmodulin | W‐7 (25 µM) | – |
| Src, MAPK, FAK, and PI3 kinases | Inhibitor | Inhibits ICOS‐L Downmodulation a |
|---|---|---|
| Src Tyr Kinases Fyn, Hck, Lck | PP2 (5 µM) | – c |
| MEK‐1 MAPK | U0126 (2.5 µM) | – b |
| P38 MAPK | SB203580 (1 µM) | –b |
| Focal Adhesion Kinase (FAK) | FAK Inhibitor I (10 µM) | –c |
| Focal Adhesion Kinase (FAK) | FAK Inhibitor II (10 µM) | – c |
| PI3‐K p110α | A66 (1 µM) | – c |
| PI3‐K p110δ | IC87114 (1 µM) | – c |
| PI3‐K p110α+δ | ETP‐46321 (1 µM) | – c |
| PI3‐K (All) | LY294002 (10 µM) | –c |
M12 ICOS‐L GFP cells were incubated with ICOS+ D10 cells. ICOS‐L staining in control assays of M12 ICOS‐L GFP cells incubated with medium or with H4–.A5 ICOS– D10 mutant cells was taken as a reference.
a
Surface ICOS‐L staining enhanced by 0–10% (–), 10–25% (+), 25–50% (++), 50–75% (+++), or > 75% (++++) of ICOS‐L levels in M12 ICOS‐L GFP cells incubated with medium or with H4–.A5 ICOS– D10 mutant cells.
b
Incubation 15 min.
c
Incubation 60 min, fixed ICOS+ cells.