Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 1. Expression analysis of matriptase, HAI-1, HAI-2, and prostasin in B cancer cells by reverse- transcription/qPCR (a,b), western blotting (c–e), and flow cytometry (f). (a) Bar graph of relative mRNA expression levels of matriptase (Mat), HAI-1, HAI-2, prostasin (Pro) in Daudi (n = 4), Namalwa (n = 5), Ramos (n = 3), Raji (n = 3), JeKo-1 (n = 3), and RS4;11 cells (n = 2) using GAPDH as the reference. The prostasin bars do not appear in the bar graph, as the actual qPCR readouts were registered as “N/A” by the instrument. (b) Bar graph of mRNA quantity ratio of HAI-2 to matriptase after normalization with the GAPDH level in each cell line in (a). (c) Western blotting images of matriptase (Ab: A300-221A), HAI-2, and GAPDH. Twenty micrograms of total protein from the cell lysate of each individual culture (including 2 repeats) were analyzed. Daudi, lanes 1–3; Namalwa, lanes 4–6; Ramos, lanes 7–9. Top panel, matriptase (Mat); middle panel, HAI-2; bottom panel, GAPDH. (d) Densitometry bar graph of relative protein quantities of matriptase and HAI-2 using GAPDH as the reference. (e) The quantitative ratio of HAI-2 to matriptase in each cell line. (f) Flow cytometry histogram of matriptase expression evaluation in Ramos cells. The Ramos cells (4 × 105) were labeled with the matriptase antibody as described in the Materials and Methods section. The matriptase-positive cells are shown in the PE-A subset (blue peak). Cells without the matriptase antibody labeling (red peak) were not detected in the PE-A subset and were used as the gating control.

Article Snippet: The human Burkitt lymphoma cell lines Daudi (ATCC® CCL-213TM), Namalwa (ATCC® CRL-1432TM), Ramos (RA 1) (ATCC® CRL-1596TM), Raji (ATCC® CCL-86TM), the JeKo-1 (ATCC® CRL-3006TM) human mantle cell lymphoma cell line, and the RS4;11 (ATCC® CRL-1432TM) human acute lymphoblastic leukemia cell line were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription, Western Blot, Cytometry, Flow Cytometry, Labeling, Antibody Labeling, Control

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 2. Prostasin exosomes reduce matriptase quantity in B cancer cells. (a) Western blot images of matriptase (Ab: A300-221A) in samples from the cell lysate (top panel) and the conditioned media (bottom panel) after incubation with prostasin exosomes (Pro) or exosomes without prostasin (KO). The Daudi cells (2 × 105 cells each) were incubated with the exosomes in 50 µL of OPTI-MEM I/2%FBS (lanes 1–4) or RPMI medium (lanes 5–8) overnight. One-half of each cell lysate or 40 µL of each media supernatant were analyzed. (b) Western blot images of GAPDH from (a). (c) Densitometry of relative intensities of matriptase in the cell lysate or media (d). Data presented are the average intensity of lanes 1, 3, 5, 7 versus that of lanes 2, 4, 6, 8 after normalization with GAPDH in (b). (e) Western blot images of matriptase (top panel; Ab: sc-365482) in the Daudi, Namalwa, and Ramos cells treated with exosomes isolated from the HEK293T cells. Cells (2.5 × 105) were co-cultured with prostasin exosomes (Pexo, lanes 3, 6, 9) or vector exosomes (Vexo, lanes 2, 5, 8) in 100 µL of OPTI-MEM I/2%FBS. Cells without exosomes (None, lanes 1, 4, 7) were cultured in the same conditions. Bottom, GAPDH western blot image. (f) Bar graph of (e) expressed as the relative intensities of matriptase

Article Snippet: The human Burkitt lymphoma cell lines Daudi (ATCC® CCL-213TM), Namalwa (ATCC® CRL-1432TM), Ramos (RA 1) (ATCC® CRL-1596TM), Raji (ATCC® CCL-86TM), the JeKo-1 (ATCC® CRL-3006TM) human mantle cell lymphoma cell line, and the RS4;11 (ATCC® CRL-1432TM) human acute lymphoblastic leukemia cell line were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Western Blot, Incubation, Isolation, Cell Culture, Plasmid Preparation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 3. B cell matriptase quantity reduction by wild-type prostasin. (a) Western blot images of matriptase (Ab: sc-365482) and GAPDH in the Daudi (top two panels), Ramos (middle two panels), and Namalwa (bottom two panels) cells treated with exosomes isolated from the Calu-3 cells and sublines with over-expressed prostasin or variants. Calu-3, parent cells; KO, subline with prostasin

Article Snippet: The human Burkitt lymphoma cell lines Daudi (ATCC® CCL-213TM), Namalwa (ATCC® CRL-1432TM), Ramos (RA 1) (ATCC® CRL-1596TM), Raji (ATCC® CCL-86TM), the JeKo-1 (ATCC® CRL-3006TM) human mantle cell lymphoma cell line, and the RS4;11 (ATCC® CRL-1432TM) human acute lymphoblastic leukemia cell line were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Western Blot, Isolation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 4. Trypsin-like serine protease activity measurement. (a) Line graphs of trypsin-like serine protease activity expressed as relative fluorescent units (RFU). After mixing 2 × 106 cells with exosomes (Vexo or Pexo) or with RPMI medium alone in a total volume of 500 µL for 24 h, the activity in the conditioned medium was measured continually for 120 min. (b) Activity graph of Vexo or Pexo exosomes from the HEK293T-Vec or Pro cells in RPMI medium without cells, used as background controls. (c) Bar graph of data from (a) after subtracting the background controls of (b). * denotes p < 0.05. (d) Dot plots of trypsin-like serine protease activity in media collected 10 days after cell–exosome co-culturing. a.u., arbitrary units. Data were analyzed in GraphPad Prism 9. ANOVA, p < 0.05 for Daudi, Namalwa, and Ramos, p > 0.05 for RS4.

Article Snippet: The human Burkitt lymphoma cell lines Daudi (ATCC® CCL-213TM), Namalwa (ATCC® CRL-1432TM), Ramos (RA 1) (ATCC® CRL-1596TM), Raji (ATCC® CCL-86TM), the JeKo-1 (ATCC® CRL-3006TM) human mantle cell lymphoma cell line, and the RS4;11 (ATCC® CRL-1432TM) human acute lymphoblastic leukemia cell line were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Activity Assay

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 5. Ectopic expression of prostasin in B cancer cells. (a) Western blot analysis of transient expression of prostasin (P) or vector alone (V) in the Daudi, Namalwa, and Ramos cells. The lysate from 2 × 105 cells of each type was analyzed. Top panel, matriptase (Ab: sc-365482); middle panel, prostasin; bottom panel, GAPDH. (b) Flow cytometry analysis of Namalwa sublines with tetracycline-induced prostasin expression or vector alone. Red peak (vector-alone cells) and sky-blue peak (prostasin-expressing cells) are samples without the prostasin antibody incubation. Orange peak (vector-alone cells) and green peak (prostasin-expressing cells) are samples incubated with the prostasin antibody. All samples were incubated with a secondary antibody conjugated with the fluorophore Cy3, and 10,000 cells of each sample were analyzed in a CytoFLEX S flow cytometer. The data were analyzed with FlowJo™software v10.8.1 and are presented in the histogram. (c) Western blot analysis of NamalwaTR sublines. One hundred thousand cells of each sample were analyzed. Lanes 1 and 4 or V, samples of the vector control subline; lanes 2 and 5 or P, samples of the subline with the wild-type prostasin; lanes 3 and 6 or M, samples of the subline with a serine active-site mutant prostasin. Left panel, cells were grown in OPTI-MEM I/2%FBS with 1 µg/mL tetracycline (with tet); right panel, cells were grown without tetracycline (no tet) for 8 days. Top two panels, matriptase antibody (sc-365482); bottom two panels, prostasin antibody. (d) Western blot analysis of tet-conditioned media from (c). Two hundred milliliters of the conditioned media were precipitated with trichloroacetic acid (TCA) (final 16.7%) at 4 ◦C overnight. The pellet was collected via centrifu- gation and analyzed. The membrane was blotted with the AF3946 human matriptase/ST14 catalytic domain antibody.

Article Snippet: The human Burkitt lymphoma cell lines Daudi (ATCC® CCL-213TM), Namalwa (ATCC® CRL-1432TM), Ramos (RA 1) (ATCC® CRL-1596TM), Raji (ATCC® CCL-86TM), the JeKo-1 (ATCC® CRL-3006TM) human mantle cell lymphoma cell line, and the RS4;11 (ATCC® CRL-1432TM) human acute lymphoblastic leukemia cell line were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Plasmid Preparation, Flow Cytometry, Incubation, Cytometry, Software, Control, Mutagenesis, Membrane