JCS027201 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Time-course of pea3 and erm expression knock-down. Relative mRNA expression levels of pea3 in si pea3 1 (dark grey) and erm in si erm 2 (light-colored grey), versus si ctrl transfected MMT cells (dark), assessed by semi-quantitative RT-PCR, 24, 48 and 96 hours post transfection. Results are expressed as ratios of pea3 or erm to cyclophilin mRNA levels (1=si ctrl). (B) pea3 and erm comparative mRNA expression in ‘normal’ mammary epithelial cells (TAC) and MMT cells. Relative mRNA expression level of pea3 and erm in TAC and MMT cells assessed by quantitative RT-PCR. Results are expressed as ratios of pea3 or erm to cyclophilin mRNA levels. Data are the mean of triplicate ± s.d. (C) Pea3 protein comparative expression in ‘normal’ mammary epithelial cells (TAC) and MMT cells. 80 µg of total protein extracts of MMT and TAC cells were analyzed by western blot using anti-Pea3 and control anti-Actin antibodies. In these experimental conditions, Pea3 is not detectable in the TAC cells.

• Supplemental Figure S2 -

  Fig. S2. (A) Validation of shRNA-mediated pea3 expression knock-down. Relative mRNA expression level of pea3 in MMT cells infected with pRS-pea3 B versus control pRS retroviruses, as assessed by quantitative RT-PCR. Results are expressed as ratios of pea3 to cyclophilin mRNA levels (1=Rs) and are the mean ± s.d. of three experiments in duplicate. (B) Proliferation and migration assays. MMT cells infected with pRS-pea3 A-B versus control pRS retroviruses were counted 72 hours post-spreading (proliferation) or after 18 hours of migration through a microporated membrane (migration). Data are the mean of triplicate ± s.d. of a representative experiment and correspond to a percentage of repression compared to the control Rs cells. (C) Anchorage-independent growth assay. MMT cells infected with pRS-pea3 A, pRS-pea3/erm versus control pRS retroviruses were mixed with agar 0.65% and cultivated during 15 days. Experiments were done three times. Data are the mean ± s.d. of triplicate of a representative experiment.

• Supplemental Figure S3 -

  Fig. S3. Significantly regulated Pea3 target genes. Annotation of the 130 genes that are significantly (fold change >2, P<0.01) down- or up-regulated when comparing MMT cells transfected with si pea3 1 and si ctrl siRNAs. Analysis was performed using proprietary software (see Materials and Methods). For subtraction profiles data were NeONORM (Noth et al., 2006a) normalized with the free parameter k set to 0.2. Logarithmic (base two) fold changes and ANOVA statistical analysis were performed according to standard methods. The probe to gene and probe to pathway annotation was done according to (Noth and Benecke, 2005), and is based on the combined GO/Kegg/Panther annotations.

• Supplemental Figure S4 -

  Fig. S4. Significantly regulated Erm target genes. Annotation of the 117 genes that are significantly (fold change >1.5 P<0.02) down- or upregulated when comparing MMT cells transfected with si erm 2 and si ctrl siRNAs. Details as described in Fig. S3.

• Supplemental Figure S5 -

  Fig. S5. Significantly regulated Pea3 and Erm target genes. Annotation of the ten genes that are regulated by both Pea3 and Erm factors. Details as described in Fig. S3.

• Supplemental Table S1 -

  Table S1. Significantly regulated Pea3 target genes. Table listing of the significantly (logQ>1.0, P<0.01) regulated Pea3 target genes in logQ decreasing order is shown. Gene expression profiles between MMT cells transfected with si pea3 1 and MMT cells transfected with si ctrl, as well as mock-transfected cells were compared.

• Supplemental Table S2 -

  Table S2. Significantly regulated Erm target genes. Table listing of the significantly (fold change >1.5, P<0.02) regulated Erm target genes in logQ decreasing order is shown. Gene expression profiles between MMT cells transfected with si erm 2 and MMT cells transfected with si ctrl, as well as mock-transfected cells were compared.

• Supplemental Table S3 -

  Table S3. Significantly regulated Pea3 and Erm target genes. Table listing of the significantly ten regulated target genes common to Pea3 and Erm, in logQ decreasing order is shown.