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cmi12173-sup-0001-si.doc2750K

Fig. S1. (related to Fig. 1). Dominant-negative Dia1 expression inhibits FHOD1-induced stress fibre formation.

A. Expression of a constitutively active derivative of FHOD1 (FHOD1ΔDAD, green) induces actin stress fibre (red) formation in HeLa cells (upper panels). Coexpression of a dominant-negative derivative of Dia1 (F1F2Δ1, white) inhibits FHOD1 induced stress fibre formation (bottom panels).

B. The per cent of transfected cells with increased stress fibre formation was determined by immunofluorescence. N = 3, error bars represent SEM.

C. HeLa cells were transfected with control or FHOD1 siRNA. Twenty-four hours post siRNA transfection, cells were transfected with FHOD1-GFP, where indicated. Lysates were probed with anti-FHOD1 antibody to detect the presence of FHOD1-GFP and endogenous FHOD1.

D. Cells were transfected with either control or FHOD1 siRNA. Twenty-four hours post transfection, cells were infected with S. Typhimurium for 10 min. Cells were then fixed and immunostained for endogenous FHOD1, S. Typhimurium and phalloidin. The same exposure settings were used when imaging endogenous FHOD1 in control and FHOD1 siRNA-treated cells.

E. Western blot analysis was used to confirm knock-down of FHOD1 or Arp3. Cells were transfected with indicated siRNA 48h prior to lysis. Cell lysates were prepared as described in Experimental procedures, and separated on an 8% SDS-PAGE gel. Lysates were probed with antibody recognizing endogenous FHOD1 or Arp3. Antibody against GAPDH was used to confirm equal loading.

Fig. S2. (related to Fig. 6). FHOD1-3A-SR-HA is not phosphorylated during S. Typhimurium invasion.

A. Cells were transfected with control or FHOD1 siRNA. Twenty-four hours post siRNA transfection, cells were transfected with the indicated expression plasmid (FHOD1-HA or FHOD1-SR-HA). Lysates were probed with anti-HA and equal loading was validated with antibody against β-tubulin.

B. Cells were first transfected with the indicated expression plasmid and subsequently infected with S. Typhimurium for 20 min. Uninfected cells were used as a control. Lysates were probed with phosphoFHOD1 (Thr1141) antibody and equal loading was validated with antibody against HA.

Fig. S3. (related to Fig. 8). Knock-down of ROCK isoforms. Cells were transfected with indicated siRNA 48h prior to lysis and lysates were prepared as described in Experimental procedures. Lysates were probed with antibody against endogenous ROCK I or ROCK II. Equal loading was confirmed with antibody against β-tubulin.

Fig. S4. (related to Fig. 9). RhoA is necessary for FHOD1 phosphorylation during S. Typhimurium invasion.

A. Cells were co-transfected with FHOD1-HA and RhoA siRNA. Invasion with S. Typhimurium was allowed to proceed for 20 min and cell lysates were prepared as described in Experimental procedures. Lysates were probed with anti-phosphoFHOD1 (Thr1141) and HA blotting validated equal loading.

B. HeLa cells were co-transfected with FHOD1-HA and active RhoA. Cell lysates were prepared as described in Experimental procedures, and separated on an 8% gel. Lysates were probed with an antibody against phospho-FHOD1. Expression of the CFP-tagged active Rho GTPase was confirmed by probing with an antibody against GFP. Total FHOD1 protein was determined with an antibody against HA. Calyculin A treatment was used as a positive control. Cells were co-transfected with FHOD1-HA and eGFP. Prior to lysis, cells were treated with 50 mM Calyculin A.

C. Cells were transfected with FHOD1-GFP and infected with the indicated S. Typhimurium mutant for 10 min. Phalloidin was used to visualize actin-rich invasion ruffles. Three independent experiments were performed and 100 invasion ruffles were scored for the presence or absence of FHOD1-GFP. Statistical analysis was performed with one-way anova and post-hoc Dunnett's test. **P < 0.01 and ***P < 0.001.

cmi12173-sup-0002-mov1.mov8037K

Movie S1. Recruitment of FHOD1-GFP and Arp3-mCherry to invasion site (related to Fig. 1) Cells were transfected with FHOD1-GFP and Arp3-mCherry and infected with NHS-647 labelled S. Typhimurium. Movies were taken with Quorum WaveFX-X1 spinning disc confocal system and Volocity Software. Four Z-stacks were taken, and movies are extended projections of all stacks. Live imaging microscopy was carried out for 50 min and 24 continuous frames are shown.

cmi12173-sup-0003-mov2.mov13861K

Movie S2. Three-dimensional (3D) reconstruction of FHOD1 and Arp3 (related to Fig. 2B). Images were taken with Delta Vision microscope and 3D compilation was completed with Volocity Software. Z-stacks were taken 0.2 μm apart. FHOD1 (red) and Arp3 (green) localize to distinct regions in the S. Typhimurium invasion site.

cmi12173-sup-0004-mov3.mov3733K

Movie S3. Formation of invasion ruffle in control cells (related to Fig. 4A). Cells were transfected with control siRNA and infected with RFP-expressing S. Typhimurium. Movies were taken with Quorum WaveFX-X1 spinning disc confocal system and Volocity Software. Four Z-stacks were taken, and movies are extended projections of all stacks. Live imaging was carried out for 35 min and 23 continuous frames are shown.

cmi12173-sup-0005-mov4.mov2759K

Movie S4. Formation of invasion ruffle in Arp3 knock-down cells (related to Fig. 4A). Cells were transfected with siRNA targeting Arp3 and infected with RFP-expressing S. Typhimurium. Movies were taken with Quorum WaveFX-X1 spinning disc confocal system and Volocity Software. Four Z-stacks were taken, and movies are extended projections of all stacks. Live imaging was carried out for 46 min and 22 continuous frames are shown.

cmi12173-sup-0006-mov5.mov3274K

Movie S5. Formation of invasion ruffle in FHOD1 knock-down cells (related to Fig. 4A). Cells were transfected with siRNA targeting FHOD1 and infected with RFP-expressing S. Typhimurium. Movies were taken with Quorum WaveFX-X1 spinning disc confocal system and Volocity Software. Four Z-stacks were taken, and movies are extended projections of all stacks. Live imaging was carried out for 43 min and 22 continuous frames are shown.

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