Supplemental Figure 1. Comparative histology of footpad skin prepared from a
transgenic mouse with a negative littermate showing normal skin cell layer
composition. Transgenic positive hMGFP mouse (A) and negative littermate (B)
footpad skin was removed from euthanized animals. Vertical cross sections were fixed in
acetone for 2 min and haematoxylin & eosin (H&E) stained, dehydrated through graded
concentrations of ethanol and mounted in Histomount. The scale bar is 20 µm.
Supplemental Figure 2. CBL3 siRNA potently and specifically inhibits pre-existing
hMGFP expression in mouse epidermis. Transgenic hMGFP mouse footpads were
treated daily (intradermal injection) with 60 µg of CBL3 or non-specific control
K6a_513a.12 siRNA for 14 days as described in Fig. 4. The mice were sacrificed and
skin sections (10 µm) prepared. hMGFP expression (or lack thereof) was visualized by
fluorescence microscopy. A, C, E and G. Cross sections from mouse footpad
intradermally injected with the non-specific siRNA (K6a_513a.12). B, D, F and H.
Cross sections from mouse footpad treated with CBL3 siRNA. C and D were coinjected
with 5 µg of pUC19. Scale bar is 20 µm. Nuclei are visualized with DAPI stain (blue).
This figure is an expanded version of Fig. 4.
Supplemental Figure 3. CBL3 siRNA does not induce IL-6 or TNF mRNA
expression in cultured human peripheral blood mononuclear cells. Freshly-isolated
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PBMCs were transfected with siRNA/DOTAP (50 pmoles/4.2 µg) or poly I:C-ss/DOTAP
(3.0 µg/1.5 µg). RNA was isolated from PBMCs at the indicated times and reverse
transcribed using random hexamer primers. The resulting cDNA was analyzed by qPCR
for IL-6 (left panel) and TNF (right panel) levels relative to GAPDH (determined in
triplicate). Commonly, siRNA-mediated immunostimulation of cytokine proteins in
freshly-prepared PBMCs is measured 16 to 24 h after treatment19,43,44. However, recent
data suggest that the cytokine response to lipidic siRNA formulations is transient and
short-lived, peaking between 2 – 8 h and resolved within 24 h after treatment16,20. The 2
– 8 h timeframe for maximal cytokine expression is consistent with our qPCR results for
TNF and IL-6 following poly I:C treatment as cytokine mRNA levels return to near
baseline. A qPCR assay for interferon alpha (Applied Biosystems Hs00855471_g1) was
also performed but no signal was detected (data not shown); it should be noted that no
positive control was run to ensure the primer/probe set was working properly. For
additional timepoints, see Supplemental Figure 4.
Supplemental Figure 4. Extended timecourse of IL-6 and TNF mRNA expression in
siRNA-treated cultured human peripheral blood mononuclear cells. Freshly-isolated
PBMCs were transfected with siRNA/DOTAP (50 pmoles/4.2 µg), poly I:C-ss (Sigma #
P1032)/DOTAP (6 µg/4 µg) or poly I:C-ds (Sigma # P9582)/DOTAP (6 µg/4 µg). RNA
was isolated from untreated PBMCs (N/A) or cells treated with the indicated nucleic
acids for 0.5, 2, 4, 8, 16, 24 and 36 h and reverse transcribed using random hexamer
primers. The resulting cDNA was analyzed by qPCR as described in Materials and
Methods for IL-6 (left panel) and TNF (right panel) levels relative to GAPDH
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(determined in triplicate). Minor induction of IL-6 and TNF mRNA levels are observed
with siRNA treatment as compared to both the single- and double-stranded poly I:C, and
of the three siRNAs analyzed, CBL3 induces the lowest levels of IL-6 and TNF mRNA.
It should be noted that treatment with either single- or double-stranded poly I:C shows
peak induction of both IL-6 and TNF mRNA earlier than treatment with siRNA. IL-6
induction peaks at 4 to 8 h when treated with the poly I:C controls, while siRNAmediated induction peaks at 16 h. Similarly, treatment with the poly I:C controls results
in peak TNF induction at 4 h, while treatment with siRNA results in peak induction at ~8
to 16 h. A qPCR assay for IFN-alpha (Applied Biosystems Hs00855471_g1) was also
performed, but little or no signal was detected (data not shown).
Supplemental Video File 1. Video of Tg CBL/hMGFP mouse footpad treated with
irrelevant siRNA (K6a_513a.12) and imaged using DAC fluorescence microscopy.
This is a 2-D image stack video of the corresponding 3-D image in Fig. 6 created with
Amira software. 2-D en face images are translated in depth from the surface to 100 µm
deep.
Supplemental Video File 2. Video of Tg CBL/hMGFP mouse footpad treated with
CBL3 siRNA and imaged using DAC fluorescence microscopy.
This is a 2-D image
stack video of the corresponding 3-D image in Fig. 6 created with Amira software. 2-D
en face images are translated in depth from the surface to 100 µm deep.
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