Supplementary Figures and Tables
Fig. S1. IMT504 does not alter heat nocifensive responses in SNI-injured rats. Acute heat pain was evaluated using the tail immersion test in naïve (gray columns), SNI+VEH (black columns), SNI+PolyC (brown column) and SNI+IMT504 (blue columns), 1 day before injury and 3 and 21 days after treatment. Data represents an n = 6 per group and are expressed as mean ± SEM. Statistical significance was established by means of two- way ANOVA followed by the Bonferroni post-hoc test: P> 0.05.
Fig. S2. IMT504 promotes the mobilization and infiltration of MSCs into the ipsilateral sciatic nerve of SNI rats. (A-F) Flow cytometry gating strategy to identify MSCs (CD45-CD90+CD29+ cells) in sciatic nerve (A and D), peripheral blood (B and E) and bone marrow (C and F) from naïve (A-F upper), SNI+VEH (A-F center) or SNI+IMT504 (A-F bottom) rats, 3 (A-C) or 21 (D-F) days after treatment. (A-F left) Dot- plots show representative distribution of CD45- cells. (A-F right) Dot-plots show representative signal distribution of CD90+CD29+ cells identified from CD45- pre-selected cells. (G) Flow cytometry gating procedures to detect MSCs (CD45-CD90+CD29+) in the contralateral sciatic nerve from SNI+VEH (G upper) and SNI+IMT504 (G lower) rats, 21 days after treatment. (G left) Dot-plots show representative distribution of CD45- cells. (G right) Dot-plot show representative signal distribution of CD90+CD29+ cells identified from pre-selected cells. (H) Mean ± SEM of CD45-CD90+CD29+ cells analyzed by flow cytometry in contralateral sciatic nerves from SNI+VEH (gray column) or SNI+IMT504 (blue column) rats, 21 days after treatment. Data represents an n=5 rats per group.
Statistical significance was established by means by unpaired t-test: P> 0.05.
Fig. S3. In vitro migration assay of rat or human MSCs towards conditioned media.
Representative photomicrographs of rat (a-h) or human (i-p) untreated (a-d; i-l) or IMT504-pre-treated (m-p) MSCs (each dot represents individual DAPI-positive MSC nuclei) attached to the membrane of the Micro Chemotaxis Chamber after migration towards DMEM (a and e; i and m), or CM from naïve (b and f; j and n), or 7 day-SNI ipsilateral (c and g; k and o) or contralateral (d and h; l, p) sciatic nerves.
Fig. S4. Confocal visualization of rat or human MSCs expose to IMT504. (a-b) Photomicrographs of rat (a) and human (b) MSCs. In red, IMT504-Texas Red fluorescent signal; in green, vimentin immunofluorescent signal, and in blue DAPI fluorescent signal.
Arrowheads indicate IMT504-Texas Red-positive intracellular granules. Scale bars: 10 μm.
Sequences
Gene Sense (5'-3') Antisense (3'-5')
-actin TACCATGCCAACTTCTGTCTGGGA TACCATGCCAACTTCTGTCTGGGA Tnf- TCGTAGCAAACCACCAAGCA CCCTTGAAGAGAACCTGGGAGTA
Il-1 CACCTCTCAAGCAGAGCACAG GGGTTCCATGGTGAAGTCAAC Il-10 TACCATGCCAACTTCTGTCTGGGA TACCATGCCAACTTCTGTCTGGGA Tgf-1 TACCATGCCAACTTCTGTCTGGGA TACCATGCCAACTTCTGTCTGGGA Cxcl12 TGCATCAGTGACGGTAAGCCA ATCCACTTTAATTTCGGGTCAA
Cxcr4 TCCTGCCCACCATCTATTTTATC ATGATATGCACAGCCTTACAT
Table 1. Sequence of the primers used in the present study