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Fig. S1. MICAL2 and MICAL3 mRNA are expressed in HeLa cells. (A) cDNA was prepared from HeLa cells grown on 35-mm dishes. PCR reactions were performed on HeLa cDNA with primers against β-actin, MICAL1 and MICAL2. Amplified cDNAs were then separated on 2% agarose gels containing ethidium bromide. (B) HeLa cells grown on 35-mm dishes that were either mock-treated or treated with MICAL3-siRNA for 72 hours. PCR reactions were performed from cDNA prepared from these cells with primers specific for MICAL3 isoform1, MICAL3 isoform3 and β-actin. Amplified cDNAs were then separated on 2% agarose gels containing ethidium bromide.
Fig. S2. MICAL1 FAD overexpression affects focal adhesion plaques. HeLa cells grown on coverslips were transiently transfected with either HA-MICAL1 (A−C) or the MICAL1 FAD domain only (D−F). After 18 hours, cells were fixed, permeabilized and incubated with anti-HA antibody and anti-phospho-paxillin antibodies. Cells were then incubated with Alexa-Fluor-568-conjugated anti-mouse and Alexa-Fluor-488-conjugated anti-rabbit secondary antibodies. Scale bar: 10 µm.
Fig. S3. Prediction of MICAL1 coiled-coil breakers. The MICAL1-CC domain (residues 925−961) (A) and the MICAL1-CC domain with residues 940 and 941(AA) mutated to PP (B) were analyzed by the Paircoil program.
Fig. S4. At moderate expression levels MICAL2/MICAL1 chimeras localize to filamentous structures devoid of F-actin. HeLa cells grown on coverslips were transiently transfected with HA-MICAL1/MICAL2 (A−C). After 18 hours, cells were fixed, permeabilized and incubated with anti-HA antibody followed by Alexa-Fluor-568-conjugated anti-mouse secondary antibody and Alexa-Fluor-488-conjugated phalloidin. Scale bar: 10 µm.
Fig. S5. Effect of MICAL proteins on endocytic organelles and microtubules. (A−D) HeLa cells grown on coverslips were transiently transfected with either full-length HA-MICAL1, truncated HAMICAL1 containing only the FAD domain or full-length HA-MICAL2. After 18 hours, cells were fixed, permeabilized and incubated with anti-HA antibody along with either anti-EEA1 (A), anti-Giantin (B), anti-α-tubulin (C) or anti-γ-tubulin (D) antibodies. Then cells were washed and incubated with Alexa-Fluor-568-conjugated anti-rabbit and Alexa-Fluor-488-conjugated anti-mouse secondary antibodies. Scale bars: 10 µm.
Fig. S6. Effect of MICAL3 depletion on actin stress fiber level. HeLa cells grown on cover-slips were either mock-treated (A) or treated with MICAL3-siRNA (B) for 72 hours (see Fig. S1B for knockdown efficiency). Cells were then fixed, permeabilized and incubated with Alexa-Fluor-488-conjugated phalloidin. Scale bar: 10 µm.
Fig. S7. Ability of FAD domain of MICAL1 to produce ROS in vivo. (A−C) HeLa cells grown on coverslips were transiently transfected with Tomato-tagged MICAL1 containing only the FAD domain. After 18 hours, cells were fixed, permeabilized and incubated with Alexa-Fluor-488-conjugated phalloidin. (D−I) HeLa cells grown on Lab-Tek chambers were transiently transfected either with Tomato-tagged MICAL1 FAD domain (G−I) or its vector only (D−F). After 18 hours, live cells were treated with 10 µM of H2DCFDA and analyzed. Scale bar: 10 µm.