Takahiro Masuda, Shosuke Iwamoto, Ryohei Yoshinaga, Hidetoshi Tozaki-Saitoh, Akira

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1 upplementary nformati Trancripti factor drive 2X4 reactive microglia gating neuropathic pain Takahiro Mauda, houke wamoto, yohei Yohinaga, Hidetohi Tozaki-aitoh, Akira Nihiyama, Tak W. Mak, Tomohiko Tamura, Makoto Tuda, Kazuhide noue upplementary igure 1-15 and reference

2 a b c Microarray data kda 5 ene pilateral (EM) alateral (EM) pi/a tudent t tet rf (6.3) (6.5) 1.3 >.5 rf (77.4) (169.2). >.5 -antiene rf (3.) 19.6 (6.).94 >.5 rf (7.73) (3.64) 1.44 <.1 rf (12.) (5.4) 1.36 =.16 -ene WT rf5-/- pi pi upplementary igure 1. Microglia-pecific upregulati following N. (a) Exprei of family ancripti factor in the pinal cord 7 day after N by three or four independent DNA microarray analye. The data of three member (4, 6 and 7) were excluded from the tatitic becaue of a detecti p-value of greater than.1 (n=3 or 4; tudent t tet). ha already been identified a a upregulated gene in the ipilateral pinal cord after N. (b) n itu hybridizati (H) analyi of rf5 mna in the pinal doral horn of WT mice 7 day after N. Arrowhead indicate rf5 mna ignal. (cale bar, 3 μm) (c) Wetern blot analyi of and β-ac protein in the pinal cord of WT and rf5 / mice 3 day after N. pi, ipilateral;, calateral. ull-ize blot are hown in upplementary ig. 13. a ol b 15 rf5 mna (relative) 1 5 l1b mna (relative) 1..5 Tnfa mna (relative) rx4 mna (relative) rx7 mna (relative) 1..5 t mna (relative) 1..5 upplementary igure 2. Ectopic exprei of itelf did not activate ancripti of gene in microglia. eal-time analyi of mna of (a) rf5 and (b) microglial gene in BV2 cell anduced with - or ale (col) (n=3). Value are mean ±.e.m..

3 a kda Whole cell Nuclear ytoolic ol L N-γ LaminB α-tublin b ntenity ratio ( / ) 1..5 Whole ntenity ratio ( / LaminB) 1..5 Nuclear ntenity ratio ( / α-tublin) 1..5 ytool ol L N-γ upplementary igure 3. L, or N-γ did not induce anlocati of into the nucleu in microglia. (a) Expreial diibuti analyi of protein in microglial BV2 cell eated with L (1 ng/ml), (5 μ ) and N-γ (1 U/ml) for 4 h. (b) A hitogram of the relative band intenity ratio of (normalied to, lamin B, or α-tublin) to col cell (n=4). Value are mean ±.e.m.. ull-ize blot are hown in upplementary ig. 14. a b ol hna c hna rf5 mna (relative) 1..5 <.1 2rx4 mna (relative) 1..5 rf5 mna (relative) ibrectin ol hna hna upplementary igure 4. Knockdown of microglial exprei by lentiviral-mediated anducti of hna. (a, b) eal-time analyi of rf5 (a) and 2rx4 (b) mna in BV2 cell anduced with a lentiviral vector encoding either hna or col hna. Value repreent the relative ratio of rf5 or 2rx4 mna (normalied to 1 mna) to the cell with col hna (n= 6, tudent t tet). (c) Exprei of rf5 mna in or col hna-anduced BV2 cell eated with fibrectin (n=6, <.1, e-way ANOVA with Bferri' multiple compari tet). Value are mean ±.e.m..

4 l1b mna (relative) Vehicle * L/N-γ l6 mna (relative) WT rf5 -/- WT rf5 -/- WT rf5 -/- Tnfa mna (relative) 25 upplementary igure 5. mpaired producti of proinflammatory cytokine in rf5 / macrophage. eal-time analyi of mna of l1b, l6 and Tnfa in WT and rf5 / periteal macrophage eated with or without L (1 ng/μl) / N-γ (1 unit/ml) for 4h. Value repreent the relative ratio of l1b, l6 or Tnfa mna (normalied to the value for 1 mna] to WT col cell (vehicle) (n =4, *<.5, <.1, e-way ANOVA with Tukey' multiple compari tet). Value are mean ±.e.m.. a b c kda 5 ol NA ntenity ratio (relative to ol) ina2 =.96 ol NA 2. ina2 WT (g) 1..5 ol NA ina2 =.69 ina inj. re 5 7 Day after N kda pilateral 5 Naive Time after A inj. (d) alateral 5 Naive Time after A inj. (d) upplementary igure 6. N-induced going exprei in the pinal cord i required for tactile allodynia. (a) Upper, repreentative immunoblot of and protein in homogenate obtained from the pinal cord of mice eated with col and ina-2 day 7 pot-n. Lower, a hitogram of the relative band denity ratio of (normalied to ) to the col NA-eated mice (n=6, tudent t tet). (b) everal of N-induced tactile allodynia by inathecal adminiati of ina-2 (2 pmol) ce a day for 2 day (5 and 6 day pot-n) in WT mice (n=7, tudent t tet). (c) epreentative wetern blot analyi of protein (of three experiment) in the ipilateral and calateral pinal cord of WT mice before (Naive) and after inaplantar A injecti. Value are the mean ±.e.m.. ull-ize blot are hown in upplementary ig. 15.

5 rf5 mna (relative) 1..5 Aif1 mna (relative) 1..5 l1b mna (relative) ### L oly(:) ol hna hna L oly(:) ol hna hna L oly(:) ol hna hna upplementary igure 7. oly(:)- or L-induced gene exprei in micorglia are ly modetly changed by knockdown of. eal-time analyi of mna of rf5, Aif1 and l1b in col or rf5 hna-anduced BV2 cell 6 h after eatment of oly(:) (5 μg/ml) or L (1 ng/ml). Value repreent the relative ratio of mna (normalied to the value for 1 mna] to col hna-anduced cell (n =6; <.1, ### <.1 v. ol hna (L), e-way ANOVA with Tukey' multiple compari tet). Value are mean ±.e.m..

6 pilateral alateral pilateral alateral Naive Naive Time after N (day) Time after N (day) Naive Naive Time after N (day) Time after N (day) upplementary igure. Unedited full blot of igure 1d. Whole cell igure 2a ibrectin ol hna hna ibrectin ol hna hna Nuclear 75kDa LaminB ibrectin ol hna hna ibrectin ol hna hna ibrectin ol hna hna ytoolic α-tublin ibrectin ol hna hna igure 2d 2X4 - - ibrectin - ol hna hna - - ibrectin - ol hna hna upplementary igure 9. Unedited full blot of igure 2.

7 o nt ro l (l ow) (h ig h) o nt ro l (K 79 E) o nt ro l (K 79 E) igure 3d o nt ro l (lo w ) (h ig h) igure 3c WT igure 3j rf-/wt pi pi rf-/- pi pi upplementary igure 1. Unedited full blot of igure 3. - rl- l- 5 l- rl- 5t t h h h h h h igure 4a - - rl- l- 5 l- rl- 5t t h h h h h h igure 4c igure 4e 2X4 - rl- l- 5 l- rl- 5t t h h h h h h l l- l- l- l l- l l h h h h h h h h h h h h - rl- l- 5 l- rl- 5t t h h h h h h l- 5- l- rl- 5- rl- rl l ll t t t h h h h h h h h h h h h upplementary igure 11. Unedited full blot of igure 4.

8 ol NA ina1 ol NA ina1 upplementary igure 12. Unedited full blot of igure 6c. WT rf5-/wt rf5-/- pi pi pi pi L ol Whole cell L ol upplementary igure 13. Unedited full blot of upplementary igure 1c. L ol 75kDa LaminB L ol Nuclear α-tublin L ol L ol ytoolic upplementary igure 14. Unedited full blot of igure 3a.

9 upplementary igure 6a ol NA ina2 ol NA ina2 upplementary igure 6c pilateral alateral Naive Naive Time after A inj. (d) Time after A inj. (d) pilateral alateral Naive Naive Time after A inj. (d) Time after A inj. (d) upplementary igure 15. Unedited full blot of upplementary igure 6. upplementary eference 1. Mauda T, et al. i a critical ancripti factor for anforming microglia into a reactive phenotype. ell ep 1, (212).