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. Author manuscript; available in PMC: 2024 May 9.
Published in final edited form as: Immunity. 2023 Apr 27;56(5):998–1012.e8. doi: 10.1016/j.immuni.2023.04.001

Fig. 4. In SLC46-transfected cells, fand anti-folates inhibit iE-DAP transport to varying degrees.

Fig. 4.

(A) HEK293T cells were transiently transfected with empty vector (EV), Slc46a1, Slc46a2, or Slc46a3 expression plasmids, and challenged with 30 μM click-iE-DAP with or without folates/antifolates. 1 hr after the challenge, cells were fixed, and then iE-DAP entry was visualized by confocal microscopy following reaction with CalFluor488-Azide. SLC46A2 more effectively transported iE-DAP, compared to SLC46A3, while SLC46A1 was inactive. MTX (250 μM) blocked iE-DAP transport through SLC46A2 or SLC46A3 whereas another antifolate, pemetrexed (PTX, 250 μM) and folic acid (FA) (250 μM) interfered with SLC46A2 mediated transport while MDP (30 μM) more potently inhibited SLC46A3 transport.

(B) Mean fluorescence intensity computed from images similar to panel (A) across three independent replicates. Each dot in the graph represents fluorescence from a cell. N.I. indicates no inhibitor.

In panel A, representative images are shown from at least three independent experiments. Panel B uses a two-way ANOVA test and Tukey’s multiple comparisons test to determine significance. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns, not significant. The scale bar is 10μM. For inhibitor structures, see Figure S4.