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cmi12176-sup-0001-fs1.eps2087K

Fig. S1. C12 and TQ416 do not initiate inflammatory responses in bone marrow derived macrophages (BMDM). Images of arrays used to assess BMDM cytokine secretions; secreted molecules are identified by colour-coded rectangles with corresponding cytokine names listed to the right (cytokines appear in duplicate on each array). Internal controls appear in the top and bottom left corners of each array. Panels 5 and 6 indicate that TQ416 and C12 did not stimulate cytokine production (see also Fig. 3).

cmi12176-sup-0002-fs2.eps42160K

Fig. S2. Characterization of inflammatory cell infiltration in intradermal C12 injection model.

A. Panels of hematoxylin and eosin stained skin sections from different mice are shown after injection of (from top to bottom) control micelles and micelles loaded with C12, C12 plus TQ416 and TQ416. Images are shown at low magnification (top) and for an expanded region (bottom; identified by the dashed box at low magnification). Scale bars represent 100 μm (top and bottom).

B. (left) High magnification image of hematoxylin and eosin stained skin section from mouse injected with C12-loaded micelles. (right) Characteristic staining of polymorphonuclear (1) and mononuclear (2) cells; dashed boxes in the initial image identify the expanded regions. Scale bar represents 20 μm; expanded regions (right) are ∼ 12 × 30 μm.

C. Immunohistochemical analysis of cell infiltration in skin injected with C12 (left) and C12 plus TQ416 (right). In C12-injected skin (left), positive responses (brown) were observed for markers of hematopoietic cells (CD45), macrophages (F4/80) and neutrophils (Ly6G). These responses were not observed in C12 plus TQ416 injected (right) or control micelle injected skin (data not shown). Scale bar represents 50 μm.

D. Quantitative analysis of inflammatory cell infiltration to the dermis. Images show examples of original data (left) and data after binarization (middle) under control conditions (top) and after injection of C12-loaded micelles (bottom). Scale bar represents 50 μm. (right) Intensity analysis provides a distribution of pixels that contain cells (intensity of 255 for 8-bit image) and those that do not (intensity of 0) that is used to calculate relative immune cell infiltration (see also Fig. 6).

cmi12176-sup-0003-fs3.eps187K

Fig. S3. Characterization of cytoplasmic Ca2+ in wt MEFs, FRT cells and primary MEFs.

A. (left) Typical C12-mediated (black) and control (no treatment; gray) Ca2+ response in wt MEFs characterized over 15 min. (right) Pre-treatment of wt MEFs with C12 (50 μM, 20 min) prevents TG-mediated release of Ca2+ (green). For reference, representative data from cells treated with TG is shown (black).

B. Characterization of cytoplasmic Ca2+ responses in FRT cells. (top left) Ca2+ responses under control conditions (black) and after TQ416 (1 μM) treatment (red). (top right) Ca2+ responses in FRT cells stimulated with C12 (200 μM; blue) and with C12 plus TQ416 (1 μM; red). (bottom) Cytoplasmic Ca2+ changes in response to ATP (100 μM; left) and thapsigargin (TG, 1 μM; right). Traces are shown in the absence (blue) and presence of TQ416 (1 μM, red); and in TG-treated cells that were pre-treated with C12 (100 μM, 20 min; green).

C. Characterization of cytoplasmic Ca2+ responses in primary MEFs. (left) Ca2+ responses in control conditions (no treatment; black) and after TQ416 treatment (1 μM; red). (right) Ca2+ responses in primary MEF cells stimulated with C12 (50 μM; blue) and with C12 plus TQ416 (1 μM; red).

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