Journal: Arthritis and rheumatism

Article Title: Immunohistochemical analysis as a means to predict responsiveness to rituximab treatment.

doi: 10.1002/art.22967

Figure Lengend Snippet: Figure 1. B cells in peripheral blood, bone marrow, and synovium before and 12 weeks after rituximab treatment. A, CD20 and CD19 expression on CD3 lymphocytes in peripheral blood from a rheuma- toid arthritis (RA) patient (left). Numbers represent the percentages of total lymphocytes. Flow cytometric data on 24 individual RA patients are shown on the right. The broken line represents the detection limit. B, CD20 and CD19 expression on CD3 lymphocytes in bone marrow from the same RA patient (left). Flow cytometric data on 18 individual RA patients are shown on the right. C, Consecutive sections of synovium stained for cytoplasmic CD20 (CD20cy) and CD79a (top). Semiquantitative scoring of CD20cy, CD79a, and CD138 expression (0 no expression; 4 highest level of expression) in synovial specimens from 17 RA patients is shown at bottom. Values are the mean and SEM. Circles indicate outliers.

Article Snippet: The remainder of the serial sections were stained with mouse anti-human Ki-67 (clone MIB-1), mouse anti-human CD68 (clone KP1), rabbit anti-mouse Ig isotype control (code X0936) (all from Dako, Heverlee, Belgium), rabbit anti-human CD3 (clone SP7; NeoMarkers, Fremont, CA), mouse anti-human CD138 (clone B-B4; Serotec, Oxford, UK), mouse IgG1–biotin isotype control (clone J1D9; Ancell/Kordia, Leiden, The Netherlands), and purified mouse IgG1 isotype control (BD PharMingen, San Diego, CA).

Techniques: Expressing, Staining

Journal: Arthritis and rheumatism

Article Title: Immunohistochemical analysis as a means to predict responsiveness to rituximab treatment.

doi: 10.1002/art.22967

Figure Lengend Snippet: Figure 3. Differences in baseline characteristics of good responders versus those with moderate or no response, as classified according to the European League Against Rheumatism response criteria. A, Expression of specific markers for B cells (cytoplasmic CD20 [CD20cy], CD79a), plasma cells (CD138), macrophages (CD68), T cells (CD3), proliferation (Ki-67), plasma cell counts, and total inflammation. B, Circulating titers of rheumatoid arthritis–specific autoantibod- ies ACPA IgG, IgM-RF, and ACPA IgM. Values are the mean and SEM. P 0.05; # P 0.10. NS not significant (see Figure 2 for other definitions).

Article Snippet: The remainder of the serial sections were stained with mouse anti-human Ki-67 (clone MIB-1), mouse anti-human CD68 (clone KP1), rabbit anti-mouse Ig isotype control (code X0936) (all from Dako, Heverlee, Belgium), rabbit anti-human CD3 (clone SP7; NeoMarkers, Fremont, CA), mouse anti-human CD138 (clone B-B4; Serotec, Oxford, UK), mouse IgG1–biotin isotype control (clone J1D9; Ancell/Kordia, Leiden, The Netherlands), and purified mouse IgG1 isotype control (BD PharMingen, San Diego, CA).

Techniques: Expressing, Clinical Proteomics

Journal: STAR Protocols

Article Title: Mass cytometry staining for human bone marrow clinical samples

doi: 10.1016/j.xpro.2022.101163

Figure Lengend Snippet:

Article Snippet: Anti-Human CD138/Syndecan-1 (DL-101)-150Nd—25 tests (1:100) , Fluidigm , Cat#3150012C.

Techniques: Recombinant, Saline, Red Blood Cell Lysis, Staining, Blocking Assay, Antibody Labeling, Software, Cell Counting, Cytometry

Journal: Cell

Article Title: Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment

doi: 10.1016/j.cell.2020.08.001

Figure Lengend Snippet:

Article Snippet: CD138 145Nd (DL101) , Fluidigm , Cat# 3145003B.

Techniques: Purification, Produced, Recombinant, Staining, Red Blood Cell Lysis, Saline, Blocking Assay, Labeling, Selection, Multiplexing, Amplification, Isolation, Software, Derivative Assay

Journal: PLoS ONE

Article Title: Prostaglandin E 2 Synthesizing Enzymes in Rheumatoid Arthritis B Cells and the Effects of B Cell Depleting Therapy on Enzyme Expression

doi: 10.1371/journal.pone.0016378

Figure Lengend Snippet: Frozen sections of synovial tissue showing diaminobenzidine (brown) staining of MPGES1 (A), COX-2 (B), COX-1 (C) and CD20 B cells (D), CD138 plasma cells (E) (hematoxiline counterstained). Insets represent higher power of image A and D. Original magnification 80x and 200x (insets). (F) and (G) Immunofluorescence pictures of double stained MPGES1 (red) and CD20+ B cells (F) and CD138+ plasma cells (G) (green). Magnification 400x.

Article Snippet: For immunofluorescence analysis of double stained synovial biopsy sections, samples were incubated with primary antiserum against MPGES1 and mouse monoclonal antibody against CD55 (Serotec, Oxford, UK), CD20 (Dako Cytomation) and CD138 (B-A38; Diaclone, Besancon, France), followed by addition of anti-rabbit Alexa 594 and anti-mouse Alexa 488 conjugated mouse monoclonal antibodies (both from Molecular Probes, Eugene, Oregon).

Techniques: Staining, Clinical Proteomics, Immunofluorescence

Journal: bioRxiv

Article Title: PD-L1 hi plasmablasts limit the T cell response during the acute phase of parasite infections

doi: 10.1101/2020.06.10.142067

Figure Lengend Snippet: a) Kinetics of plasmablasts (PB, CD19 int B220 low CD138 hi ), Germinal Center (GC) B cells (CD19 + CD3 − CD138 − B220 + CD38 low Bcl6 +) and CD3 + CD4 + T cells number in the spleen of T. cruzi infected mice. b) Representative flow cytometry plots and statistical analysis of the percentage of splenic PB (CD138 + CD19 int / low ) from WT (WT BCR, n=5) and MD4 (HEL-sp BCR, n=8) mice at 14 Dpi with T. cruzi . Correlation between the percentage of PB and the percentage of IgHEL B cells (non-related BCR) in the spleen from infected MD4 (white circles) or WT (black circles) mice (***p < 0.001, two tailed t Test). c) Representative flow cytometry plots and statistical analysis of the percentage of splenic PB from WT (n=9) and Bcl6 fl/fl CD4 Cre (Bcl-6 neg CD4+ T cells, n=5) mice at 14 Dpi with T. cruzi . (**p < 0.01, two tailed t Test) d) Representative Immunofluorescence of spleen sections (7um) from T. cruzi -infected C57BL/6 mice obtained at 14 Dpi, stained with anti-B220 (red), anti-CD3 (green) and anti-CD138 (blue). Magnification: x100 (left) and x400 (right) (n = 9).

Article Snippet: The following anti-mouse antibodies were used for FACS: CD19-PE (6D5), CD19-BV605 (6D5), CD3-AlexaFluor700 (17A2), CD3-APCCY7 (17A2), CD4-APC-Cy7 (GK1.5), CD8-PerCP-Cy5.5 (53-6.7), CD8-FITC (5H10-1), Streptavidin BV605, PD-1-BV421 (29F.1A12), PDL-1-Pecy7 (10F.9G2), Bcl6-PE-Dazzle594 (7D1), ICOS-AlexaFluor488 (C398.4A), CD45.1-BV605 (A20), CD11b-APCCy7 (M1/70), B220-APCCy7 (RA3-6B2), Live Dead Aqua 430 were from Biolegend; CD19-AlexaFluor700 (1D3), CD19-FITC (1D3), IgD-FITC (11-26c), CD8-PECy7 (53-6.7), TNF-APC (MP6-XT22), PD-L1-PE (MIH5) and CD-39-PerCP-eFluor710 (24DMS1) were from eBioscience; CD138-BV605 (281-2), CD138-APC (281-2), CD138-PE (281-2), CD138-Biotin, CD38-BV421 (90/CD38), CXCR5-Biotin (2G8), IFN-γ-FITC (XMG1.2), IL-4-PE (11B11), Ki-67-AlexaFluor647 (B56), Blimp-1-PE-CF594 (5E7), and CD8-BUV805 (53-6.7) were from BD Biosciences.

Techniques: Infection, Flow Cytometry, Two Tailed Test, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: PD-L1 hi plasmablasts limit the T cell response during the acute phase of parasite infections

doi: 10.1101/2020.06.10.142067

Figure Lengend Snippet: a) Representative histograms of PD-L1 or PD-L2 expression on splenic non-B cells (CD19 − CD138 − B220 − ), B cells (CD19 + CD3 − CD138 − B220 + ) from non-infected mice and T. cruzi -infected C57BL/6 mice and on plasmablasts (PB) (CD19 int B220 low CD138 hi ) from T. cruzi -infected C57BL/6 mice evaluated at 9, 15 and 22 Dpi. Statistical analysis of mean fluorescence intensity (MFI) of PD-L1 or PD-L2 on the mentioned cells analyzed at 15 Dpi. b) Representative plots of CD138 vs Blimp-1 of live single lymphocytes (gated on IgD − IgM − CD11b − CD24 − ) from the spleen and bone marrow (BM) obtained at 15 Dpi and 130 Dpi with T. cruzi , respectively. Representative histograms of PD-L1 on gated CD138 + Blimp-1 + cells. Numbers indicate the percentage of PD-L1 + cells. Bar graph shows statistical analysis of MFI of PD-L1 + on splenic PB and BM plasma cells (PC). c) 10 5 Splenocytes from T. cruzi infected mice obtained at 14 Dpi were incubated with T. cruzi antigens and cultures for 48h with media, anti-PD-1, anti-PD-L1 or anti-PD-L2. TNF and IFNγ concentrations were determined by Elisa in the culture supernatant. d) 1× 10 5 Non-B (CD19 neg CD138 neg ) cells in a suspension of total splenocytes (non-depleted) from T. cruzi infected mice obtained at 14 Dpi or depleted of B cells (Non B cells), plasmablasts (Non PB), PD-L1 hi cells (Non PD-L1 hi ) or PD-L1 low (Non PD-L1 lo ) cells were cultured with T. cruzi antigens for 48h. TNF and IFN✉ concentrations were determined by Elisa in the culture supernatant. e) Statistical analysis of MFI of PD-L1 on Non-B non-T non-PB cells (CD3 − CD19 − B220 − CD138 − ), T cells (CD3 + CD19 − B220 − ), naïve B cells (CD19 + CD3 − CD138 − B220 + IgD + CD38 + Bcl6 − ), GC B cells (CD19 + CD3 − CD138 − B220 + CD38 low Bcl6 + ) and PB (CD19 int CD138 + ) from the spleen of T. cruzi infected mice obtained at 14 Dpi, or from the spleen of P. chabaudi infected mice obtained at 10 Dpi; or CD4+ T cells, GC B cells of PB from the spleen of Arm or Cl13 LCMV infected mice obtained at 9 Dpi.*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 one -way ANOVA, Bonferroni post-test

Article Snippet: The following anti-mouse antibodies were used for FACS: CD19-PE (6D5), CD19-BV605 (6D5), CD3-AlexaFluor700 (17A2), CD3-APCCY7 (17A2), CD4-APC-Cy7 (GK1.5), CD8-PerCP-Cy5.5 (53-6.7), CD8-FITC (5H10-1), Streptavidin BV605, PD-1-BV421 (29F.1A12), PDL-1-Pecy7 (10F.9G2), Bcl6-PE-Dazzle594 (7D1), ICOS-AlexaFluor488 (C398.4A), CD45.1-BV605 (A20), CD11b-APCCy7 (M1/70), B220-APCCy7 (RA3-6B2), Live Dead Aqua 430 were from Biolegend; CD19-AlexaFluor700 (1D3), CD19-FITC (1D3), IgD-FITC (11-26c), CD8-PECy7 (53-6.7), TNF-APC (MP6-XT22), PD-L1-PE (MIH5) and CD-39-PerCP-eFluor710 (24DMS1) were from eBioscience; CD138-BV605 (281-2), CD138-APC (281-2), CD138-PE (281-2), CD138-Biotin, CD38-BV421 (90/CD38), CXCR5-Biotin (2G8), IFN-γ-FITC (XMG1.2), IL-4-PE (11B11), Ki-67-AlexaFluor647 (B56), Blimp-1-PE-CF594 (5E7), and CD8-BUV805 (53-6.7) were from BD Biosciences.

Techniques: Expressing, Infection, Fluorescence, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: bioRxiv

Article Title: PD-L1 hi plasmablasts limit the T cell response during the acute phase of parasite infections

doi: 10.1101/2020.06.10.142067

Figure Lengend Snippet: Gating strategies to determine the frequency of plasmablasts (PB), B cells and non-B cells (Non-B) in the spleen of C57BL6 mice infected with T. cruzi Y strain obtained at 14 Dpi. Splenocytes were stained with anti-CD19, -B220, -CD138 and anti-PD-L1 and depleted from plasmablasts (PB), non-B cells, and PD-L1 hi and PD-L1 low cells. Representative dot plot of: a) total splenocytes prior to the sort, b) post-sort excluding plasmablast (Non PB) and c) excluding B cells (Non-B cells). d-e) Dot plot representative of splenocytes without PD-L1 hi (Non PD-L1 hi ) and without PD-L1 low (Non PD-L1 low ) cells, respectively. The mean fluorescence intensity corresponding to PD-L1 expression on the remaining B cells is shown with the histogram.

Article Snippet: The following anti-mouse antibodies were used for FACS: CD19-PE (6D5), CD19-BV605 (6D5), CD3-AlexaFluor700 (17A2), CD3-APCCY7 (17A2), CD4-APC-Cy7 (GK1.5), CD8-PerCP-Cy5.5 (53-6.7), CD8-FITC (5H10-1), Streptavidin BV605, PD-1-BV421 (29F.1A12), PDL-1-Pecy7 (10F.9G2), Bcl6-PE-Dazzle594 (7D1), ICOS-AlexaFluor488 (C398.4A), CD45.1-BV605 (A20), CD11b-APCCy7 (M1/70), B220-APCCy7 (RA3-6B2), Live Dead Aqua 430 were from Biolegend; CD19-AlexaFluor700 (1D3), CD19-FITC (1D3), IgD-FITC (11-26c), CD8-PECy7 (53-6.7), TNF-APC (MP6-XT22), PD-L1-PE (MIH5) and CD-39-PerCP-eFluor710 (24DMS1) were from eBioscience; CD138-BV605 (281-2), CD138-APC (281-2), CD138-PE (281-2), CD138-Biotin, CD38-BV421 (90/CD38), CXCR5-Biotin (2G8), IFN-γ-FITC (XMG1.2), IL-4-PE (11B11), Ki-67-AlexaFluor647 (B56), Blimp-1-PE-CF594 (5E7), and CD8-BUV805 (53-6.7) were from BD Biosciences.

Techniques: Infection, Staining, Fluorescence, Expressing

Journal: bioRxiv

Article Title: PD-L1 hi plasmablasts limit the T cell response during the acute phase of parasite infections

doi: 10.1101/2020.06.10.142067

Figure Lengend Snippet: Representative flow cytometry plots and statistical analysis of the percentage of splenic PD-L1 + plasmablasts from T. cruzi-, P. chabaudi- or LCMV Cl13-infected mice. C57BL6 mice were infected with T. cruzi Y strain (n=5), P chabaudi (n=5) or with LCMV Cl13 or Arm (n=5) and the spleens were obtained at 14, 10 or 9 Dpi, respectively. Splenocytes were stained with anti-B220, anti-CD138 and anti-PD-L1.

Article Snippet: The following anti-mouse antibodies were used for FACS: CD19-PE (6D5), CD19-BV605 (6D5), CD3-AlexaFluor700 (17A2), CD3-APCCY7 (17A2), CD4-APC-Cy7 (GK1.5), CD8-PerCP-Cy5.5 (53-6.7), CD8-FITC (5H10-1), Streptavidin BV605, PD-1-BV421 (29F.1A12), PDL-1-Pecy7 (10F.9G2), Bcl6-PE-Dazzle594 (7D1), ICOS-AlexaFluor488 (C398.4A), CD45.1-BV605 (A20), CD11b-APCCy7 (M1/70), B220-APCCy7 (RA3-6B2), Live Dead Aqua 430 were from Biolegend; CD19-AlexaFluor700 (1D3), CD19-FITC (1D3), IgD-FITC (11-26c), CD8-PECy7 (53-6.7), TNF-APC (MP6-XT22), PD-L1-PE (MIH5) and CD-39-PerCP-eFluor710 (24DMS1) were from eBioscience; CD138-BV605 (281-2), CD138-APC (281-2), CD138-PE (281-2), CD138-Biotin, CD38-BV421 (90/CD38), CXCR5-Biotin (2G8), IFN-γ-FITC (XMG1.2), IL-4-PE (11B11), Ki-67-AlexaFluor647 (B56), Blimp-1-PE-CF594 (5E7), and CD8-BUV805 (53-6.7) were from BD Biosciences.

Techniques: Flow Cytometry, Infection, Staining

Journal: bioRxiv

Article Title: PD-L1 hi plasmablasts limit the T cell response during the acute phase of parasite infections

doi: 10.1101/2020.06.10.142067

Figure Lengend Snippet: a) Representative flow cytometry plots and statistical analysis of the percentage of splenic plasmablasts from T. cruzi-infected and from FasLpr, Sanroque and Trex1 mice. C57BL6 mice (n=4) were infected with T. cruzi Y strain and the spleens were obtained at 14 Dpi. Spleens from FasLpr (n=7), Sanroque (n=4) and Trex1 (n=3) mice were obtained at 10-12 weeks of age. Splenocytes were stained with anti-B220, anti-CD138 and anti-PD-L1. Data are representative of two independent experiments. Statistical analysis of PD-L1 expression (MFI) on plasmablasts (PB) from the spleen of T cruzi- infected mice and FasLpr, Sanroque and Trex1 mice. ***p < 0.001one-way ANOVA, Bonferroni post-test. c) Representative flow cytometry plots of the percentage of SWHEL-specific B cells identified as CD45.1 + HEL + cells and the percentage of SWHEL-specific plasmablasts CD138 + CD19 low in CD45.2 mice transferred with 30.000 HEL-specific B cells and immunized with 2×10 8 SRBC conjugated with HEL. Histograms show PD-L1 expression on plasmablasts from 14 Dpi- T. cruzi infected mice and SWHEL-specific plasmablasts (PB) evaluated at 7 days post immunization (DPI). d) Statistical analysis of PD-L1 expression (MFI) on plasmablasts from T. cruzi infected mice, SWHEL-specific (HEL + PB) and –non-specific (HEL neg PB) plasmablasts from CD45.2 mice transferred with 30.000 HEL-specific B cells and immunized with 2×10 8 SRBC conjugated with HEL, and on plasmablasts from mice immunized with SRBC (7 DPI). ****p < 0.0001 one -way ANOVA, Bonferroni post-test. e) Representative histograms of PD-L1 expression on B cells cultures with medium, T. cruzi trypomastigotes antigens (AgTpY), live T. cruzi trypomastigotes (TpY), TNF, TGFβ and IFNγ. Bar graph shows statistical analysis of PD-L1 expression increase on B cells cultured with the mentioned stimulus respect to medium.

Article Snippet: The following anti-mouse antibodies were used for FACS: CD19-PE (6D5), CD19-BV605 (6D5), CD3-AlexaFluor700 (17A2), CD3-APCCY7 (17A2), CD4-APC-Cy7 (GK1.5), CD8-PerCP-Cy5.5 (53-6.7), CD8-FITC (5H10-1), Streptavidin BV605, PD-1-BV421 (29F.1A12), PDL-1-Pecy7 (10F.9G2), Bcl6-PE-Dazzle594 (7D1), ICOS-AlexaFluor488 (C398.4A), CD45.1-BV605 (A20), CD11b-APCCy7 (M1/70), B220-APCCy7 (RA3-6B2), Live Dead Aqua 430 were from Biolegend; CD19-AlexaFluor700 (1D3), CD19-FITC (1D3), IgD-FITC (11-26c), CD8-PECy7 (53-6.7), TNF-APC (MP6-XT22), PD-L1-PE (MIH5) and CD-39-PerCP-eFluor710 (24DMS1) were from eBioscience; CD138-BV605 (281-2), CD138-APC (281-2), CD138-PE (281-2), CD138-Biotin, CD38-BV421 (90/CD38), CXCR5-Biotin (2G8), IFN-γ-FITC (XMG1.2), IL-4-PE (11B11), Ki-67-AlexaFluor647 (B56), Blimp-1-PE-CF594 (5E7), and CD8-BUV805 (53-6.7) were from BD Biosciences.

Techniques: Flow Cytometry, Infection, Staining, Expressing, Cell Culture

Journal: bioRxiv

Article Title: PD-L1 hi plasmablasts limit the T cell response during the acute phase of parasite infections

doi: 10.1101/2020.06.10.142067

Figure Lengend Snippet: Gating strategy to determine the percentage of plasma cells (CD19 − CD138 + ) and plasmablasts (CD19 low CD138 + ) and CD3 + CD19 − (non B cells), CD138 − CD3 − CD19 + (B cells) and CD38 + Bcl-6 neg CD3 − CD19 + (naïve B cells) and CD38 neg Bcl-6 + CD3 − CD19 + (GC B cells) from the spleen of T. cruzi infected mice.

Article Snippet: The following anti-mouse antibodies were used for FACS: CD19-PE (6D5), CD19-BV605 (6D5), CD3-AlexaFluor700 (17A2), CD3-APCCY7 (17A2), CD4-APC-Cy7 (GK1.5), CD8-PerCP-Cy5.5 (53-6.7), CD8-FITC (5H10-1), Streptavidin BV605, PD-1-BV421 (29F.1A12), PDL-1-Pecy7 (10F.9G2), Bcl6-PE-Dazzle594 (7D1), ICOS-AlexaFluor488 (C398.4A), CD45.1-BV605 (A20), CD11b-APCCy7 (M1/70), B220-APCCy7 (RA3-6B2), Live Dead Aqua 430 were from Biolegend; CD19-AlexaFluor700 (1D3), CD19-FITC (1D3), IgD-FITC (11-26c), CD8-PECy7 (53-6.7), TNF-APC (MP6-XT22), PD-L1-PE (MIH5) and CD-39-PerCP-eFluor710 (24DMS1) were from eBioscience; CD138-BV605 (281-2), CD138-APC (281-2), CD138-PE (281-2), CD138-Biotin, CD38-BV421 (90/CD38), CXCR5-Biotin (2G8), IFN-γ-FITC (XMG1.2), IL-4-PE (11B11), Ki-67-AlexaFluor647 (B56), Blimp-1-PE-CF594 (5E7), and CD8-BUV805 (53-6.7) were from BD Biosciences.

Techniques: Infection

Journal: Journal of Hematology & Oncology

Article Title: Blocking of Transient Receptor Potential Vanilloid 1 (TRPV1) promotes terminal mitophagy in multiple myeloma, disturbing calcium homeostasis and targeting ubiquitin pathway and bortezomib-induced unfolded protein response

doi: 10.1186/s13045-020-00993-0

Figure Lengend Snippet: TRPV1 expression and the effect of TRPV1 inhibition on MM cell viability in MM BM samples and MM cell lines. a TRPV1 mRNA levels in bone marrow (BM) samples from healthy donors, MM patients and MM cell lines, measured by quantitative RT-PCR. b Effect of TRPV1 antagonist AMG9810 on viability of MM cell lines and primary CD138+ cells. Cells were incubated with increasing doses of AMG9810 for 48 h, and viability was measured by the XTT method. c – e Time course of the increase in AMG9810-induced vesicle acidification, mitochondrial depolarization and cell death. RPMI8226 and CAG MM cells were exposed to AMG9810 (10 µM) for indicated time points. c Vesicle acidification was measured by flow cytometry using acridine orange (AO) dye. Representative flow cytometry histograms from RPMI8226 cells (left side). Graphs showing percentages of AO-positive RPMI8226 and CAG cells obtained from representative experiment out of three repeats (right side). d Mitochondrial depolarization (Δ Ψm loss) in AMG9810-treated cells detected by DiOC6 staining and flow cytometry analysis. Representative flow cytometry histograms from RPMI8226 cells (left side) and graphs showing percentages of MM cells with Δ Ψm loss obtained from representative experiment out of three repeats (right side). e AMG9810-induced cell death detected by PI staining. Representative flow cytometry histograms from RPMI8226 cells (left side) and graphs showing the percentages of PI-positive dead RPMI8226 and CAG cells from representative experiment out of three repeats (right side). Data are presented as mean of triplicates ± SD (** p < 0.01). f Western blot analysis of anti-apoptotic proteins BCL-XL and MCL-1, ER stress marker CHOP and mTOR pathway target pS6 in RPMI826 and CAG MM cells before and after treatment with various doses of AMG9810 for 24 h. β-Actin was used as internal control. Representative data from at least two independent experiments are shown. g Caspase 3 activation in response to AMG9810 treatment (5–20 µM, 24-h exposure) was assessed using CaspGLOW Red staining and flow cytometry analysis. Representative histograms (upper panel) and graphs showing percentages of cells with activated Caspase 3 (lower panel). Data are presented as mean of triplicates ± SD (** p < 0.01)

Article Snippet: In primary MM samples, counterstaining with anti-CD138 fluorescein (FITC)-conjugated antibody (IQ products, Netherlands) was performed.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Incubation, Flow Cytometry, Staining, Western Blot, Marker, Activation Assay

Journal: Journal of Hematology & Oncology

Article Title: Blocking of Transient Receptor Potential Vanilloid 1 (TRPV1) promotes terminal mitophagy in multiple myeloma, disturbing calcium homeostasis and targeting ubiquitin pathway and bortezomib-induced unfolded protein response

doi: 10.1186/s13045-020-00993-0

Figure Lengend Snippet: AMG9810 suppresses CXCR4 expression and activity in MM cells. Effect of AMG9810 treatment (10 µM, 1 h, 37 °C) on cell surface CXCR4 expression in RPMI8226, RPMI8226-CXCR4, OPM-1 and primary CD138 + MM cells, measured by flow cytometry. a Representative histograms demonstrating CXCR4 levels in non-treated and AMG9810-treated cells. b CXCR4 expression level is shown as mean fluorescent intensity (MFI), data are presented as mean of triplicates ± SD (** p < 0.01). c RMI8226-CXCR4 cells were pre-treated with indicated doses of AMG9810 for 30 min, and their migratory response to CXCL12 (200 ng/ml) was measured using the trans-well migration assay. d Effect of pre-treatment with AMG9810 (10 µM for 1 h at 37 °C) on CXCL12-induced (200 ng/ml, 30-min activation) intra-cellular signaling in RPMI8226-CXCR4 and OPM-1 cells. Levels of phosphorylated Erk1/2 and AKT proteins, measured by Western blot, β-actin was used as internal control. Representative data from at least two independent experiments are shown. e , f Effect of capsaicin treatment (10 µM, 1 h, 37 °C) on cell surface CXCR4 expression in RPMI8226, RPMI8226-CXCR4 and OPM-1 cells, measured by flow cytometry. g RMI8226-CXCR4 cells were pre-treated with indicated doses of capsaicin (10 µM) for 30 min, and their migratory response to CXCL12 (200 ng/ml) was measured using the trans-well migration assay. h CFSE-prelabeled RPMI8226 and RPMI8226-CXCR4 cells were pre-treated with AMG9810 (10 µM) or capsaicin (10 µM) for 30 min and then co-incubated with BMSC monolayer during 30 min; non-adherent cells were removed, and adherent CFSE-positive cells were enumerated using flow cytometry and expressed as percent of total input cells. Data are presented as mean of triplicates ± SD (** p < 0.01)

Article Snippet: In primary MM samples, counterstaining with anti-CD138 fluorescein (FITC)-conjugated antibody (IQ products, Netherlands) was performed.

Techniques: Expressing, Activity Assay, Flow Cytometry, Migration, Activation Assay, Western Blot, Incubation