JCS03431 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Effect of HDAC inhibitors on tubulin and other proteins. (A) Acetylated tubulin (Ac-tubulin), histone (Ac-histones) and total tubulin expression in cell lysates of NIH-3T3 treated with HDAC inhibitors. (B) Acetylated (Ac-Hsp90) and total Hsp90 following immunoprecipitation of Hsp90 from WT MEFs treated with HDAC inhibitors. Anti-Hsp90 from Assay Designs (Ann Arbor, MI) was used to immuneprecipitate total Hsp90, whereas anti-Ac lysine antibody (made from chemically acetylated keyhole limpet hemocyanin) was used to detect acetylation levels of all proteins. All drug treatments lasted 4 hours.

• Supplemental Figure 2 -

  Fig. S2. siRNA-expressing HDAC6 KD cells. (A) Micrographs of A549 pSuper cells expressing control plasmid (left) and A549 HDAC6 KD cells (right), immunostained for acetylated tubulin. Bar, 50 μm. (B) Acetylated (Ac-) and total tubulin expression, and (C) HDAC6 expression, in A549 pSuper and HDAC6 KD cells, determined via western blots of cell lysates. (D) Transwell chemotactic invasion assay of A549 pSuper, HDAC6 KD, and taxol-treated pSuper cells. (E) Focal adhesion area in A549 pSuper and A549 HDAC6 KD cells. *, conditions statistically different from control (P<0.05).

• Supplemental Figure 3 -

  Fig. S3. MT dynamics are decreased in the presence of HDAC inhibitors. (A)Percentage of time MTs spent polymerizing (growing) and depolymerizing (shrinking) in cells subjected to 30-minute treatments as indicated. (B) Catastrophe frequencies and (C) rescue frequencies of MTs in cells subjected to the above treatment. (D) Catastrophe frequencies and (E) rescue frequencies of MTs from WT and HDAC6 MEFs expressing 3xGFP-EMTB. HDAC6 rescue denotes HDAC6 KO MEFs transiently transfected with WT HDAC6. *, conditions statistically different from control (P<0.05).

• Supplemental Figure 4 -

  Fig. S4. Hyperacetylation protects MTs against nocodazole-induced depolymerization. (A) Micrographs of MT arrays that remained in TC-7 cells following treatment with nocodazole for the indicated times. Bar, 20 μm. (B-D) Quantification of MTs protected from nocodazole depolymerization are shown in (B) TC-7 and (C) NIH-3T3 cells treated with inhibitors, and (D) A549 HDAC6 KD and control (pSup) cells. Slopes of TSA- and taxol-treated cells in B and C were significantly different from control (DMSO) cells (P< 0.05)

• Supplemental Figure 5 -

  Fig. S5. MT acetylation protects against end-mediated depolymerization induced by ATP. (A,B) Decreased (A) rate and (B) percentage of time of end-mediated depolymerization of cytoskeletons prepared from cells treated with TSA and taxol. (C) Micrographs depicting typical MT cytoskeletons following ATP-induced depolymerization for the indicated times. Bar, 20 μm. (D) End-mediated depolymerization of MT cytoskeletons, following treatment with TSA or taxol as indicated. Slopes were significantly different (P<0.05) from control for both drug treatments. (E) Rate and (F) percentage time of MT depolymerization, after treatment with NaB; results of NaB-treated cytoskeletons were not statistically different from DMSO control (P>0.2).

• Movie 1 -

  Movie 1. Fluorescence recovery of GFP-paxillin-labeled adhesions after photobleaching in WT MEFs. This Movie corresponds to Fig. 4A,B.

• Movie 2 -

  Movie 2. Fluorescence recovery of GFP-paxillin-labeled adhesions after photobleaching in HDAC6 KO MEFs. This Movie corresponds to Fig. 4A,B.

• Movie 3 -

  Movie 3. Time-lapse images of GFP-paxillin-labeled adhesions in WT MEFs. This Movie corresponds to Fig. 4C,D.

• Movie 4 -

  Movie 4. Time-lapse images of GFP-paxillin-labelled adhesions in HDAC6 KO MEFs. This Movie corresponds to Fig. 4C,D.

• Movie 5 -

  Movie 5. Time-lapse images of MTs labeled with 3xGFP-EMTB as described in Materials and methods. This Movie corresponds to Fig. 5A-E.