1. Analysis of Signal Transducer and Activator of Transcription 3 (Stat 3) Pathway in Multiple Myeloma 
Leticia Quintanilla-Martinez, Marcus Kremer, Katja Specht, Julia Calzada-Wack, Michaela Nathrath, Robert Schaich, Heinz Höfler, Falko Fend 
The American Journal of Pathology 
Volume 162, Issue 5, Pages (May 2003)
DOI: /S (10)
Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
Western blot analysis for Stat 3, Stat 3P, cyclin D1, and anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2 in human MM cell lines. Each lane contains 60 μg of protein extract from the following cell lines: lane 1, Granta 519; lane 2, KMS-20; lane 3, U266; lane 4, KMS-18; lane 5, KMS-5; lane 6, KMS-11; lane 7, KMM1; lane 8, KMS-12; lane 9, OPM2. Lane 1 (Granta 519, mantle cell lymphoma cell line) and lane 8 (KMS12) represent the t(11;14) translocated cell lines. Expression of cyclin D1 is limited to these two cell lines. Lane 3 (U266) represents the cell line with known constitutive activation of Stat 3. Western blot analysis with the N-terminal anti-Stat 3 antibody demonstrates both Stat 3α and Stat 3β (92 and 83 kd, respectively) in all cell lines. In contrast only KMS20 and U266 show strong positivity for Stat 3 phosphorylated. Bcl-xL and Mcl-1 expression is present in all cell lines with no apparent correlation between these proteins and the presence of phosphorylated Stat 3. Bcl-2 is negative in three of the cell lines (KMS20, KMS5, and KMM1). Note that the two cell lines with expression of cyclin D1 show high levels of Bcl-2.
The American Journal of Pathology  , DOI: ( /S (10) )
Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
Immunohistochemical analysis of Stat 3 and Stat 3P. A: KMS18 cell line block used as control. The tumor cells show cytoplasmic positivity for Stat 3 without nuclear staining. Inset: Note that the cells are completely negative when stained with the Stat 3P antibody. B: U266 cell line block used as control. The tumor cells show a strong, crisp nuclear positivity for Stat 3P without cytoplasmic staining. Inset: Note that by immunostaining with the Stat 3 antibody, the cells show strong nuclear and cytoplasmic staining. C–F: Primary MM cases stained for Stat 3 and Stat 3P. C: Case 2, group 1. The tumor cells show cytoplasmic and nuclear positivity for Stat 3. D: The same case immunostained with the Stat 3P antibody reveals a strong nuclear expression confirming the presence of activated Stat 3. E: Case 36, group 2. The vast majority of tumor cells show cytoplasmic expression of Stat 3 indicating the presence of steady-state Stat 3. Note the lack of nuclear staining. F: The same case is negative for anti-Stat 3P. Note the positive nuclear staining of the endothelial cells used as internal control. Immunoperoxidase staining; original magnifications, ×400.
The American Journal of Pathology  , DOI: ( /S (10) )
Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
Immunohistochemical analysis of cyclin D1 in primary MM cases. A: The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++) (case 29, group 2). B: MM with lymphoplasmacytoid features (case 32, group 2). The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++). C: MM case with nuclear positivity in 20 to 50% of tumor cells (++) and co-expression of Stat 3P (case 20, group 1). D: MM with nuclear positivity in 20 to 50% of tumor cells (++) (case 35, group 2). Note that the intensity of the staining varies from cell to cell. E: MM with nuclear positivity in 10 to 20% of tumor cells (+) and expression of Stat 3P (case 19, group 1). F: MM negative for cyclin D1. Note the presence of rare, positive plasma cells (arrows) (case 46, group 3). Immunoperoxidase; original magnifications: ×200 (B); ×400 (A, C–F).
The American Journal of Pathology  , DOI: ( /S (10) )
Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
Immunohistochemical analysis of anti-apoptotic proteins in MM cases. A: Mcl-1 expression in a MM case with activated Stat 3 (case 17, group 1). Note the strong cytoplasmic staining. B: Mcl-1 expression in a MM case with cyclin D1 overexpression. Note the weaker cytoplasmic positivity in comparison with the previous case and with the small reactive lymphocytes (arrows) (case 34, group 2). C and D: Bcl-2 expression in MM cases. C: Bcl-2 is strongly expressed in the cytoplasm in the vast majority of tumor cells. D: An example of a MM case negative for Bcl-2. Note the Bcl-2 positivity of a reactive lymphocyte used as internal control. E and F: Bcl-xL expression in MM cases. E: Bcl-xL is positive in the cytoplasm of the tumor cells. F: MM with lack of expression of Bcl-xL. Note the positivity in the cytoplasm of reactive histiocytes used as internal control. Immunoperoxidase; original magnifications, ×400.
The American Journal of Pathology  , DOI: ( /S (10) )
Copyright © 2003 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
Comparison of proliferation rate in Bcl-2-positive and Bcl-2-negative cases. Statistical analysis was done using the Mann-Whitney U-test. Cases that are Bcl-2-positive have a mean proliferation rate of 10%, whereas cases with Bcl-2-negative have a mean proliferation rate of 30% (P = ).
The American Journal of Pathology  , DOI: ( /S (10) )
Copyright © 2003 American Society for Investigative Pathology Terms and Conditions