Article Figures & Data
Figures
- Figure 1.
A, TMS1 expression in primary DCIS and invasive ductal carcinomas. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded breast tumor tissue samples. The TMS1 antibody was an affinity-purified rabbit polyclonal antibody (EU107) raised against a peptide corresponding to amino acids 182 to 195 of human TMS1, and was used at a dilution of 1:400 as described ( 18, 19). Immunocomplexes were detected by the avidin-biotin complex method, using diaminobenzidine as the chromogen. Slides were counterstained with hematoxylin (blue). i to iii, TMS1 expression in DCIS. Note the unusual staining pattern in DCIS lesions with retention of TMS1 expression in the cells closest to the myoepithelium and basement membrane and loss of expression in the more dysplastic cells filling the luminal space. This pattern was most often observed in solid-form DCIS (i and ii) and cribriform DCIS (iii) lesions. iv to vi, TMS1 expression in invasive ductal carcinomas. Whereas some infiltrating glands retain TMS1 expression (iv and v), 3 of 19 invasive carcinomas showed complete loss of TMS1 expression (vi). Note that even in ductal carcinoma cases that lose TMS1 expression (vi; ca), surrounding normal inflammatory cells (iv and vi; inf) retain TMS1 expression. B, relationship between TMS1 and E-cadherin expression in primary DCIS. Parallel sections were stained with H&E (i) or analyzed for the expression of TMS1 (ii) or E-cadherin (iii). The TMS1 antibody (EU107) was used at a dilution of 1:400, and the E-cadherin antibody (Invitrogen #15068) was used at a dilution of 1:50. Note the unusual TMS1 staining pattern in DCIS lesions with retention of TMS1 expression in the outermost cells and loss of expression in the more dysplastic cells filling the luminal space. The same cells that lose TMS1 expression retain E-cadherin expression. iv to vi, higher power (×600) magnifications of the same lesion shown in i to iii (×100). Shown is a representative DCIS lesion from a single individual. An independent example from a second individual is shown in Supplementary Fig. S1.
- Figure 2.
TMS1 protein and mRNA are induced during anoikis. A and B, expression of TMS1 after detachment. MCF10A cells were suspended in growth medium containing 0.5% methylcellulose and plated onto poly-HEMA coated plates, and harvested at the indicated time points. A, protein lysates were subjected to western blot analysis using antibodies against TMS1, Bim, and GAPDH. B, total cellular RNA was isolated and TMS1 mRNA expression levels were quantified by reverse transcription and real-time PCR analysis as previously described ( 13). Relative starting quantities were determined by comparison to MCF7 cDNA standard curve included in each run. Shown is the relative increase in TMS1 mRNA compared with time zero after normalization to an 18s rRNA internal control. Columns, mean of three independent experiments performed in triplicate; bars, SD. C and D, detachment-induced up-regulation of TMS1 is not dependent on NF-κB signaling. C, MCF10A cells were infected with 20 multiplicity of infection of a dominant-negative IκBα adenoviral construct the night before plating onto poly-HEMA plates. Cells were left attached (A) or plated on poly-HEMA–coated dishes (S) for 24 h. Protein lysates were harvested and subjected to western blot analysis with the indicated antibodies. D, top panel, MCF10A cells were transfected with either 200 nmol/L scrambled siRNA (control) or siRNA targeting the p65 subunit of NF-κB (p65). After 48 h, cells were suspended in growth medium containing 0.5% methylcellulose and plated onto poly-HEMA–coated 10-cm plates. Lysates were collected and analyzed by Western blot with antibodies to TMS1, the p65 subunit of NF-κB, and GAPDH. D, bottom panel, total cellular RNA was isolated and TMS1 mRNA expression levels were determined and analyzed by real-time PCR as described in B. Shown is the relative expression of TMS1 mRNA compared with time zero after normalization to an 18s rRNA internal control. Columns, mean of three independent experiments performed in triplicate; bars, SD.
- Figure 3.
Knockdown of TMS1 expression inhibits anoikis. A, MCF10A cells were transfected with 200 nmol/L of scrambled siRNA (control) or siRNA#1 targeting TMS1 (TMS1). After 36 h, cells were plated on poly-HEMA–coated dishes, and protein was isolated at timed intervals and subjected to western analysis with the indicated antibodies. B, MCF10A cells were transfected as in A. After 36 h, cells were plated on poly-HEMA–coated dishes. The following day, the cells were harvested and analyzed for apoptosis using the DNA fragmentation cell death detection ELISA. A portion of the cell sample was retained, harvested for protein, and subjected to western blot analysis for TMS1 and β-tubulin (inset).
- Figure 4.
Impact of TMS1 knockdown on ERK signaling and Bim up-regulation during anoikis. A, detachment-induced up-regulation of Bim requires TMS1. MCF10A cells were transfected with 50 nmol/L of scrambled siRNA (control) or siRNA#1 targeting TMS1 (TMS1). Left, after 48 h, cells were suspended in growth medium containing 0.5% methylcellulose and plated onto poly-HEMA–coated plates. Left, lysates were collected at the indicated time points and subjected to Western blot analysis using the indicated antibodies. Right, Bim mRNA expression levels were quantified by real-time PCR analysis. Relative starting quantities were determined by comparison to MCF7 cDNA standard curve included in each run. Shown is the increase in Bim mRNA relative to time zero after normalization to an 18s rRNA control. Columns, mean of three independent experiments assayed in triplicate; bars, SD. B, TMS1 loss promotes persistent ERK activation. MCF10A cells were transfected with 50 nmol/L scrambled siRNA or siRNA#1 targeting TMS1. After 48 h, cells were suspended in growth medium containing 0.5% methylcellulose and plated onto poly-HEMA–coated plates. Lysates were collected at the indicated time points and subjected to western blot analysis using the indicated antibodies. C and D, the requirement for TMS1 in detachment-induced Bim accumulation is apoptosis-independent. C, MCF10A cells were transfected with 200 nmol/L of scrambled siRNA or TMS1#1 siRNA. After 48 h, the cells were plated onto poly-HEMA coated plates in the absence or presence of 250 nmol/L z-VAD-FMK. Protein was harvested at the indicated time points and analyzed by western blot analysis with the indicated antibodies. D, MCF10A-pBABE and MCF10A-Bcl-2 cells were transfected with 200 nmol/L scrambled or TMS1#1 siRNA. After 48 h, cells were suspended in growth medium containing 0.5% methylcellulose and plated onto poly-HEMA–coated plates. Lysates were collected at the indicated time points and subjected to Western blot analysis with the indicated antibodies.
Volume 69, Issue 5
