Supplemental Material to: 

PRECLINICAL STUDY 

α‐Parvin, a pseudopodial constituent, promotes cell motility and is associated with 

lymph node metastasis of lobular breast carcinoma 

in Breast Cancer Research and Treatment 

 

Masaoki Ito, Man Hagiyama, Takahiro Mimae, Takao Inoue, Takashi Kato, Azusa 

Yoneshige, Jun Nakanishi, Tadashi Kondo, Morihito Okada, and Akihiko Ito 

 

Correspondence to: 

Dr Akihiko Ito, Department of Pathology, Faculty of Medicine, Kinki University, 377‐2 

Ohno‐Higashi, Osaka‐Sayama, Osaka 589‐8511, Japan; aito@med.kindai.ac.jp 

Tel: +81 72 366 0221; Fax: +81 72 360 2028 

 

   

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Table of Contents 

Supplemental Fig. S1. Identification of α‐parvin by 2D‐DIGE and LS‐MS/MS 

Supplemental Fig. S2. Western blotting of α‐parvin in MDA‐MB‐231 cells 

Supplemental Fig. S3. Immunohistochemistry of IDC for α‐parvin 

Supplemental Table S1. IDC patient characteristics   

Supplemental Table S2. Lymph node metastasis of ILC 

Supplemental Table S3. Lymphatic invasion of ILC 

 

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Supplemental Fig. S1. 

A 

 

 

 

 

 

 

 

B

3

C

Spot No.

Accession No. a

protein ID a Identified protein a

pI (cal) b MW (cal) (D) b

Protein score c

Peptide matches

Sequence coverage (%) d

1 Q9NVD7 PARVA_HUMAN Alpha-parvin 5.69 42274 218 3 11.8

Ion charge state (+)

MZ (obs) e Mass (obs) f Mass (calc) g

Delta h Miss i Mascot ion score j

Peptide sequence

2 765.8276 1529.6406 1528.8097 0.831 0 86.69 LNVAEVTQSEIAQK

821.1122 1640.2098 1639.8934 0.3165 0 66.57 VLIDWINDVLVGER

873.8647 1745.7148 1744.8591 0.8557 0 68.29 QIQEEITGNTEALSGR

a Accession numbers of proteins, protein ID, and protein name are derived from Swiss-Prot and NCBI non-redundant databases b Theoretical isoelectric point and molecular weight obtained from Swiss-Prot and the ExPASy database (http://au.expasy.org) c Mascot score for the identified proteins based on the peptide ions score (P < 0.05) (http://www.matrixscience.com)

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d Percentage of identified amino acids e Experimental m/z value f Observed relative molecular mass g Calculated relative molecular mass h Difference (error) between the experimental and calculated masses i Number of missed cleavage sites j Mascot ion score (http://www.matrixscience.com/search_form_select.html)

 

Supplemental Fig. S1. Identification of α‐parvin by 2D‐DIGE and LS‐MS/MS (A) A protein spot of interest (#1, indicated by arrows) detected by 2D‐DIGE of pseudopodial (left) and whole‐cell (right) extracts from MDA‐MB‐231 cells. (B) LC‐MS/MS spectra for protein spot #1. Three peptide sequences were identified. (C) Results of the Mascot database search of the identified peptide sequences. 

 

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Supplemental Fig. S2. 

A   

 

 

 

 

 

 

B 

 

 

 

 

Supplemental Fig. S2. Western blotting of α‐parvin in MDA‐MB‐231 cells (A) MDA‐MB‐231 cells were transfected with control siRNA, α‐parvin‐targeting siRNAs (#1 and #2), or α‐parvin cDNA. Cell lysates were examined by western blotting using antibodies against α‐parvin (ab139270) or phosphorylated α‐parvin (ab79409). Reprobing with an anti‐β‐actin antibody indicates protein load per lane. (B) Densitometric analysis of the western blot results shown in A. Band intensities were converted to densitometric values, and the protein levels of α‐parvin and phosphorylated α‐parvin relative to β‐actin were calculated. The means of 3 independent experiments were plotted with bars indicating SD. * and ** P < 0.01 and 0.05 by Mann‐Whitney U test vs. untransfected cells  

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Supplemental Fig. S3. 

A        B       Supplemental Fig. S3. Immunohistochemistry of IDC for α‐parvin Adjacent sections of formalin‐fixed, paraffin‐embedded IDCs were stained with H&E (left) and an anti‐α‐parvin antibody (HPA005964) (red‐brown; middle and right). Nuclei were counterstained with hematoxylin (pale blue). Two representative α‐parvin‐negative tumors are shown. A, solid‐tubular type; B, scirrhous type. Boxed areas in the middle panels are enlarged in the left. Bar, 100 µm in the left and middle panels; 20 µm in the right 

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Supplemental Table S1. IDC patient characteristics 

Variables Total (n = 21)

Age (range, median) 40–86, 64

Histological variant, n (%) Papillo-tubular 7 (33) Solid-tubular 7 (33)

Scirrhous 7 (33)

Lymph node metastasis, n (%) Positive 9 (43) Negative 12 (57)

Lymphatic invasion, n (%) Positive 8 (38) Negative 13 (62)

Vascular invasion, n (%) Positive 1 (5) Negative 20 (95)

pT status,a n (%) pT1 12 (57) pT2 9 (43) pT3 0 (0)

Nuclear atypia,b n (%) Score 1 1 (5) Score 2 13 (62) Score 3 7 (33)

Mitotic count,b n (%) Score 1 7 (33) Score 2 8 (38) Score 3 6 (29)

Estrogen receptor, n (%) Positive 16 (76) Negative 5 (24)

Progesterone receptor, n (%) Positive 11 (52) Negative 10 (48)

HER2,c n (%) Positive 5 (24)

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Negative 16 (76)

Triple negative,d n (%) Yes 3 (14) No 18 (86)

a According to TNM classification (ref. 22 in main text) b According to WHO classification (ref. 1 in main text) c Human epidermal growth factor receptor 2 d Negative for estrogen receptor, progesterone receptor, and HER2

 

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Supplemental Table S2. Lymph node metastasis of ILC 

Univariate analysis 

Lymph node metastasis Variables Positive Negative P value

Histological variant Classic 8 17 Alveolar 3 6

S

olid 11 11 0.6 2 2

α-parvin Positive 14 7 Negative 8 27 0.003

Lymphatic invasion Positive 12 2 Negative 10 32 < 0.001

Vascular invasion Positive 0 0 Negative 22 34 1.000

pT status a pT1 11 18 pT2 10 15 pT3 1 1 0.909

Nuclear atypia b Score 1 9 11 Score 2 11 18

S

core 3 2 5 0.939

Mitotic count b Score 1 19 30

Score 2 1 4 Score 3 2 0 0.534

Estrogen receptor Positive 20 30 Negative 2 4 0.899

Progesterone receptor Positive 16 24 Negative 6 10 0.897

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HER2 c Positive 2 3 Negative 20 31 0.656

Triple negative d Yes 2 2 No 20 32 0.940

a According to TNM classification (ref. 22 in main text) b According to WHO classification (ref. 1 in main text) c Human epidermal growth factor receptor 2 d Negative for estrogen receptor, progesterone receptor, and HER2

 

Multivariate analysis 

Variables Odds ratio 95% CI e P value

α-parvin (Positive vs. Negative) 5.153 1.301–20.412 0.020 Lymphatic invasion (Positive vs. Negative) 15.344 2.700–87.209 0.002

e Confidence interval 

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Supplemental Table S3. Lymphatic invasion of ILC 

Univariate analysis 

Lymphatic invasion Variables Positive Negative P value

Histological variant Classic 3 22

Alveolar 2 7 S

olid 9 13 0.147

α-parvin Positive 9 12 Negative 5 30 0.0 83 3

Lymph node metastasis Positive 12 10 Negative 2 32 < 0.001

Vascular invasion Positive 0 0 Negative 14 42 1.000

pT status a pT1 4 25 pT2 9 16 pT3 1 1 0.291

Nuclear atypia b Score 1 7 13 Score 2 6 23 Score 3 1 6 0.687

Mitotic count b Score 1 12 37 Score 2 1 4

S

core 3 1 1 0.964

Estrogen receptor Positive 12 38 Negative 2 4 1.000

Progesterone receptor Positive 9 31 Negative 5 11 0.733

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HER2 c Positive 1 4 Negative 13 38 0.787

Triple negative d Yes 2 2 No 12 40 0.549

a According to TNM classification (ref. 22 in main text) b According to WHO classification (ref. 1 in main text) c Human epidermal growth factor receptor 2 d Negative for estrogen receptor, progesterone receptor, and HER2

Multivariate analysis 

Odds ratio 95% CI e P value

α-parvin (Positive vs Negative) 1.804 0.391–8.327 0.450 Lymph node metastasis (Positive vs Negative)

15.334 2.699–87.122 0.002

e Confidence interval. 

             

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