(A) Schematic illustration of sciatic nerve ligation. P, proximal; D, distal to the ligation site.

Transcription

1 SUPPLEMENTRY INFORMTION SUPPLEMENTL FIGURES Figure S1. () Schematic illustration of sciatic nerve ligation. P, proximal; D, distal to the ligation site. () Western blot of ligated and unligated sciatic nerves segments from 4 individual mice. (C) Western blot of ligated and unligated proximal optic nerves segments 4 individual mice. (D) Western blot of ligated and unligated proximal optic nerves 48 hours after injury. (E) Mouse spinal cord was injured by hemisection and dissected after 24 hours. Injured and uninjured spinal cord longitudinal sections were stained with the indicated antibodies. cetylated tubulin is detected following injury (white arrow). ar, 200µm. (F) 3.5mm-long longitudinal sections of sciatic nerves were stained with the indicated antibodies. White dashed arrow indicated the ligation site. ar, 500µm. (G) The intensity of acetylated and tyrosinated tubulin was normalized to α-tubulin along the 3.5mm-long longitudinal. Figure S2. Raw data plots and linear fitted lines of the normalized intensity of acetylated tubulin over α- tubulin as a function of distance. Figure S3. () Experimental scheme and representative images of spot cultured DRG neurons. ar, 500µm. () Quantification of maximum radial length (n=11 for vehicle, n=13 for scriptaid, mean ± SEM, ***p<0.001). 1

2 Figure S4. () Cell bodies of GFP-expressing DRG neurons were fixed following the in vitro regeneration assay. ar, 400µm. () GFP intensity in axons after axotomy at 0 hour (left panel). The GFP fluorescence intensity of a square area (2.7 x 0.1mm) at 0.1mm proximal to the axotomy line was measured both vehicle and scriptaid conditions (right panel, n=8 for each, mean ± SEM). Red dotted line indicates axotomy line. ar, 200µm. (C) The GFP fluorescence intensity in uninjured axons in both vehicle and scriptaid conditions was quantified (right panel, n=8 for each, mean ± SEM). ar, 200µm. Figure S5. () Representative image illustrating loss of YFP fluorescence at the crush site spanning ~1mm. ar, 200µm. () Representative longitudinal sections of crushed sciatic nerve stained with GP-43 and SMI hours after crush. ar, 200µm. (C) Quantification of normalized GP-43 intensity. GP-43 intensity was plotted in function of the distance from crush line (white dotted line in, n=3 for each, mean ± SEM). Figure S6. () Uninjured axons of control, HDC4, HDC5 or HDC6 knock down-drg neurons. () Raw data plots of the normalized intensity of acetylated tubulin over α-tubulin in function of distance. (C) Western blot analysis of DRG infected with, HDC4, HDC5 or HDC6 lentivirus. (D) Western blot analysis of DRG infected with or shhdc6 lentivirus. HDC5 does not affect the levels of HDC6, and vice versa, HDC6 knock down does not affect HDC5. (E) Representative western blot image and quantification of HDC5 phosphorylation level following a 2 hour sciatic nerve ligation (n=7, *p<0.05, mean ± SEM). 2

3 Figure S7. () HDC1, HDC2 or HDC3 were knocked down prior to axotomy and DRG neurons stained with the indicated antibodies. ar, 100µm. () verage intensity plot of normalized acetylated tubulin (n=6 for each, mean ± SEM). (C) The average of absolute values of slopes of acetylated tubulin intensity over a µµ distance: shhdc1, -x, 1.3 ± 0.5(x10-4 ), +x, 24.4 ± 2.6(x10-4 ); shhdc2, -x, 1.6 ± 0.8(x10-4 ), +x, 26.4 ± 0.9(x10-4 ); shhdc3, -x, 3.5 ± 1.6(x10-4 ), +x, 28.9 ± 5.7(x10-4 ) (***p<0.001, mean ± SEM). Figure S8. () Endogenous HDC5 was knocked down and HDC5 levels were rescued by coinfecting DRG neurons with an shrn resistant human HDC5 (GFP-hHDC5). xotomized neurons were stained with the indicated antibodies. () verage intensity plot of normalized acetylated tubulin. and is same plot as figure 5. (n=7, mean ± SEM). (C) The average of absolute values of slopes of acetylated tubulin intensity over a µm distance:, 19.9 ± 2.1(x10-4 );, 5.0 ± 1.4(x10-4 ); +GFP-hHDC5, 18.1 ± 2.4(x10-4 ) (***p<0.001, mean ± SEM). (D) Western blot analysis of DRG neurons following HDC5 knock down or GFP-hHDC5 expression. Figure S9. () Experimental scheme of the in vitro tubulin deacetylase (TDC) assay. FLG-HDCs were immunoprecipitated from HEK293T cells and incubated with sciatic nerve extracts. The supernatants and the precipitants were analyzed by western blot. () In vitro TDC assay with FLG-HDC5, in the presence or absence of 5µM scriptaid. (C) Quantification of 3

4 remaining level of acetylated tubulin (n=5, **p<0.01, mean ± SEM). (D) HDC6 does not coimmunoprecipitate with FLG-HDC5. (E) Experimental scheme of in vitro TDC assay with PM stimulation of HEK293T cells prior to immunoprecipitation and CIP treatment of the immunoprecipitated HDCs. Figure S10. () The level of acetylated tubulin following HDC5 or HDC6 knock down was analyzed in cultured DRG neurons in basal, non-injured conditions.. () Quantification of (n=4, **p<0.01, mean ± SEM). Figure S11. () Time-lapse acquisitions of Fluo-4M and phase contrast images of laser-axotomized DRG neurons. ar, 10µm. () Ligated and unligated sciatic nerve were treated with EGT and analyzed by western blot with the indicated antibodies. (C) DRG neurons were axotomized and stained with p-pkc and SMI-31. ar, 100µm. ar in magnified image, 50µm. (D) Representative western blot (left panel) and quantification of p-pkcµ in ligated (L) and unligated (U) sciatic nerve (right panel, n=5, **p<0.01, mean ± SEM). (E) Raw data plots of the normalized intensity of acetylated tubulin over α-tubulin in function of distance in the presence of the PKC inhibitor Go6983. Figure S12. () PKCµ was knocked down prior to axotomy and DRG neurons stained with the indicated antibodies. ar, 100µm. () verage intensity plot of normalized acetylated tubulin (n=6 for each, mean ± SEM). (C) The average of absolute values of slopes of acetylated tubulin 4

5 intensity over a µm distance: -x, 5.5 ± 1.6(x10-4 ) (n=6), +x, 9.0 ± 5.9(x10-4 ) (mean ± SEM). (D) Raw data plots of the normalized intensity of acetylated tubulin over α-tubulin as a function of distance. (E) Western blot analysis of DRG transduced by shpkcµ lentivirus. Figure S13. () Longitudinal sciatic nerves sections were stained with TUJ1, a-tubulin and S100β. () The relative intensity of α-tubulin co-localizing with the axon specific TUJ1-positive area or with the schwann cell specific S100b-positive area were measured (n=8, mean ± SEM, ***p<0.001). SUPPLEMENTL MOVIES: Movie 1. Effect of scriptaid treatment on DRG growth cone motility and morphology. ar, 10µm. Movie 2. Effect of vehicle treatment on DRG growth cone motility and morphology. bar, 10µm. Movie 3. Calcium influx induced by laser axotomy in DRG neuron. Red dot indicates laser axotomy spot. Top to bottom; Fluo-4 raw movie, intensity profile from raw movie and DIC. bar, 10µm. Movie 4. GFP-expressing control growth cone. ar, 5µm. Movie 5. GFP-expressing HDC5 knock-down growth cone. ar, 5µm. Movie 6. GFP-expressing hhdc5 overexpressed growth cone. ar, 5µm. 5

6 DRG Ligation Sciatic nerve P D Supplemental Data C c-tub Mouse1 Mouse2 Mouse3 Mouse4 U P U P U P U P c-tub Tyr-tub Mouse1 U P D Mouse2 Mouse3 Mouse4 U P D U P D U P D D c-tub Mouse1 Mouse2 U P U P E cetylated tubulin Merge Hemisection Control F nterior G Normalized intensity of c-tub (fold) Dorsal nterior Dorsal Normalized intensity of Tyr-tub (fold) c-tub Distance from ligation site (x1mm) Tyr-tub Cho et al., Figure S1 (related to figure 1)

7 Normalized intensity of -xotomy Normalized intensity of +xotomy Normalized intensity of +xotomy w/ scriptaid Cho et al., Figure S2 (related to figure 2)

8 Plating GFP Lentivirus +Vehicle or Scriptaid Fix DIV0 DIV1 DIV3 DIV6 Vehicle Scriptaid Scale bar = 500 m Maximum radial length (mm) Vehicle *** Scriptaid Cho et al., Figure S3 (related to figure 3)

9 Vehicle Scriptaid 0 hour after axotomy Vehicle Scriptaid Fluorescence intensity (.U.) Vehicle() n.s. Scriptaid() C Vehicle Un-injured axons Scriptaid Fluorescence intensity (.U.) Vehicle n.s. Scriptaid Cho et al., Figure S4 (related to figure 4)

10 Proximal Distal 2 hours after crush GP-43 GP-43/SMI-31 C Scriptaid Vehicle Normalized GP-43 intensity (fold) Distance from crush site ( m) Cho et al., Figure S5 (related to figure 4)

11 -xotomy cetylated tubulin Merge -xotomy +xotomy shhdc6 shhdc4 C No virus shhdc4 HDC4 No virus HDC5 No virus shhdc6 HDC6 shhdc4 shhdc6 Normalized intensity (fold) (cetylated tubulin / ) Normalized intensity (fold) (cetylated tubulin / ) Normalized intensity (fold) (cetylated tubulin / ) Normalized intensity (fold) (cetylated tubulin / ) D shhdc6 HDC6 shhdc6 HDC5 E p-hdc5 HDC5 Mouse 1 Mouse 2 U L U L Normalized intensity (fold) Unligation * Ligation Cho et al., Figure S6 (related to figure 5)

12 cetylated tubulin Merge Supplemental Data shhdc1 - xotomy + xotomy verage intensity of shhdc1 -xotomy +xotomy shhdc2 - xotomy + xotomy verage intensity of shhdc2 -xotomy +xotomy shhdc3 + xotomy - xotomy C *** verage intensity of shhdc3 -xotomy +xotomy *** *** shhdc1 shhdc2 shhdc3 Cho et al., Figure S7 (related to figure 5)

13 cetylated tubulin Merge + hhdc5 D verage intensity of +GFP-hHDC5 +GFP-hHDC5 HDC5 C n.s *** *** +GFP-hHDC5 Cho et al., Figure S8 (related to figure 5)

14 FLG-HDC5 immunoprecipitated from 293T lysates Sciatic nerve extract Ppt Sup Scriptaid FLG-HDC Ppt. Sup. c-tub nti-flg C ** n.s. ** D IP : IgG nti-flg Input(10%) HDC6 Relative intensity FLG-HDC5 - FLG-HDC5 + FLG-HDC5 E FLG-HDC5 transfection HEK293T Cells were stimulated by 10 PM or vehicle Cells were lysed in uffer (-phosphatase inhibitor) or uffer (+phosphatase inhibitor) Immunoprecipitated with anti-flg antibody Magnetic beads were incubated with S or CIP Sciatic nerve extract in TDC assay buffer was incubated with prepared beads in RT eads and supernatants were separated and subjected into SDS-PGE Cho et al., Figure S9 (related to figure 5)

15 shhdc6 cetylated tubulin ** Normalized intensity (fold) shhdc6 Cho et al., Figure S10 (related to figure 6)

16 Fluo-4 Phase contrast +EGT U L c-tub Tyr-tub +55 (Sec) C p-pkc p-pkc/smi-31 Maginified D -xotomy Mouse 1 Mouse 2 U L U L p-pkc PKC Fold ** +xotomy U L xotomy E +Gö6983 Normalized intensity of -xotomy Normalized intensity of +xotomy Cho et al., Figure S11 (related to figure 7)

17 cetylated tubulin Merge PKC knock down +xotomy -xotomy * verage intensity of -xotomy (n=6) +xotomy (n=6) C -x n.s +x D PKC kcock down Normalized intensity of -xotomy Normalized intensity of +xotomy E No virus shpkc PKC Cho et al., Figure S12 (related to figure 7)

18 TUJ1 Relative intensity of (fold) *** S100 Merge xon Schwann cell Cho et al., Figure S13 (related to figure 7K)