Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: Western blot analysis of PKCδ after CRISPR-Cas9 mediated PKCδ KO. Lysates were stained for PKCδ A . or PKCδ phospho-threonine 505 (PKCδ pT505) B . Clones 6’-9 and 6’-22 were selected for further PKCδ KO studies. C . Volcano blot indicating the changes of the constitutive serine or threonine protein phosphosites of PKCδ KO compared to Ramos cells. On the X-axis the plot depicts log2 fold change of phosphorylated serine or threonine residues in the whole proteome of the two cells lines. The Y-axis shows the log10 FDR adjusted p-value. Decreased phosphorylation of serine residues of the MT pathway are shown in dark green, increase in light green. The phosphorylated serine residues of MT proteins are indicated.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Western Blot, CRISPR, Staining, Clone Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: Comparison of the expression of B cell marker protein on the surface of PKCδ KO (brown) and WT Ramos B cells (blue) by flow cytometry. The expression data of PKCδ KO are normalized to that of WT Ramos cells set to 100%. B . Volcano plot showing quantitative changes of the constitutive serine/threonine phosphorylation of substrate proteins of WT in comparison to PKCδ KO Ramos B cells. On the X-axis the plot depicts log2 fold change of phosphorylated serine or threonine residues in the whole proteome of the two cell lines. The Y-axis shows the log10 FDR adjusted p-value <0.05. The significant changes of phosphorylated CD20 serine residues are indicated and shown in orange.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Comparison, Expressing, Marker, Flow Cytometry, Phospho-proteomics

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of genes associated with destabilized MT and cytoskeleton. A. The plot shows enrichment of genes associated with positive MT disassembly in PKCδ KO compared to Ramos WT cells. Vertical black lines indicate where genes overexpressed by MT depolymerization fall in the ranked list, (p-value 0.011 and FDR 0.81). B. As indication for the disconnection from the actin-cytoskeleton to the plasma membrane the plot shows the decrease of genes essential for the localization of the portion of the cytoskeleton that lies just beneath to the plasma membrane in PKCδ KO compared to Ramos WT cells, (p-value 0.00018 and FDR 0.091). The Normalized Enrichment Score (blue line) considers the ranked list of expression differences between two independent unstimulated PKCδ KO Ramos cell clones and Ramos WT cells.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Clinical Proteomics, Membrane, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: A, B. Flow cytometry analysis of CD32b expression on the surface of CD32b KO (beige) or CD32b-tr (orange) in comparison to WT Ramos B cells (blue). The unstained control (US) is shown in light grey. C, D. Flow cytometry analysis of the expression of B cell marker protein on the surface of CD32b KO (beige) or CD32b-tr (orange) in comparison to WT Ramos B cells (blue). The expression data of CD32b KO or CD32b-tr cells are normalized to those of WT Ramos set to 100%. E. Fab-PLA study of the IgD-BCR:CD32b proximity on the surface of WT Ramos (upper left), CD32b-tr, (upper right), CD32b KO (lower left), and isolated healthy donor (HD) B cells (lower right). PLA dots in red and the nuclei stained blue by DAPI, Scale bar 5 µm, F. Quantification of Fab-PLA data with the CellProfiler and calculation of the significance between the groups with PRISM 10, one-way ANOVA, the Students t-test shows no significant difference between CD32-tr and HD B cells. Mean value are show as red bars.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Flow Cytometry, Expressing, Comparison, Control, Marker, Isolation, Staining

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: Due to the expression of the FcγRIIb receptor on Ramos, the surface abundance of critical B cell proteins more closely resembles that of naive B cells. Flow cytometry analysis shows the comparison of the expression of B cell surface protein abundance on healthy donor (HD) negative selected naïve B cells (green), Ramos (black) and FcγRIIb receptor transfected (CD32b-tr, orange) Ramos cells. Unstained (US) control in grey).

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Expressing, Flow Cytometry, Comparison, Quantitative Proteomics, Transfection, Control

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: A. Protein sequence of the N-terminal (top) and part of the C-terminal cytoplasmic tail of human CD20 (bottom). Below the WT sequence the sequence with the introduced S/A mutations (in bold letters) at the N-terminal (Nmut) and C-terminal (Cmut) cytoplasmic tail of CD20 are shown. The location of the RxxS motifs implicated in 14-3-3 binding is shown below the sequence B. Membrane topology of human CD20 with a C-terminal flag-tag (CD20-flag). The location of the two RxxS motifs is indicated with red boxes C . Flow cytometry analysis of CD32b-tr Ramos cells (orange) and reconstituted Ramos cells expressing CD20 WT (black), Nmut (green), Cmut (dark blue) or NCmut (red). The Ramos cells are tested for the expression of CD20, CD32b, IgM, IgD, and CD83. The unstained (US) CD32-tr cells are shown in grey . D. Fab-PLA study of the CD20:14-3-3 proximity in WT (upper left), Nmut (upper right), Cmut (lower left), and NCmut (lower right) Ramos B cells. PLA dots in red and the nuclei stained blue by DAPI, Scale bar 5 µm. E . Quantification of the PLA data by CellProfiler. Significant differences between groups were calculated with PRISM 10, one-way ANOVA. Students t-test shows no significant difference between CD32-tr and WT Ramos cells. F. Western blot analysis of the CD20-flag/14-3-3 association in WT, Nmut, Cmut and NCmut Ramos B cells. The amount of 14-3-3 protein in the anti-flag immunoprecipitated and total lysates is show on the left and right, respectively. Lysates were taken from at least 3 independently generated Ramos cell lines.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Sequencing, Binding Assay, Membrane, FLAG-tag, Flow Cytometry, Expressing, Staining, Western Blot, Immunoprecipitation, Generated

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: A. Schematic drawing of the domain structure of the RhoA guanine nucleotide exchange factor GEF-H1 (ARHGEF2). The functional domains are drawn as blue boxes. The location of the RRxxR motif implicated in dynein light chain binding, and the Pak2 phosphorylated S886 within the 14-3-3 binding R xx S x P motif is indicated. B . 1-PLA study of the CD20: GEF-H1 proximity in WT (upper left), Nmut (upper right), Cmut (lower left), and NCmut (lower right) Ramos B cells. PLA dots in red and the nuclei stained blue by DAPI, Scale bar 5 µm. C . Quantification of PLA of B. with CellProfiler. Significant differences between groups were calculated with PRISM 10, one-way ANOVA. Students t-test shows no significant difference between CD32-tr and WT Ramos cells. D. Western blot analysis of the constitutive CD20-flag/GEF-H1 association in WT, Nmut, Cmut and NCmut Ramos B cells. The amount of GEF-H1 protein in the anti-flag immunoprecipitated and total lysates is show on the left and right, respectively. E . Western blot analysis of free GEF-H1 in the lysates of Ramos and CD32b-tr cells as well as reconstituted Ramos cells expressing CD20 WT, Nmut, Cmut or NCmut. The amount of GEF-H1 purified with the GST-RhoAG17A beads and in the total lysate is shown at the top and bottom, respectively. F. Quantification of the Western blot data with Image Studio Lite (Licor) showing the difference of signal intensity in the immunoprecipitate (IP) versus that of the total lysates (TL).

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Functional Assay, Binding Assay, Staining, Western Blot, Immunoprecipitation, Expressing, Purification

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: 1-PLA study of A. the CD20:14-3-3, C. the CD20:GEF-H1, E. the CD20:RhoA and F. the CD20:ROCK1 proximity in CD32b-tr Ramos cells (left) and reconstituted Ramos cells expressing WT (middle) or NCmut CD20 (right). The studied Ramos cells were either untreated (top) or exposed for 5 min to RTX (bottom). PLA dots in red and the nuclei stained blue by DAPI, Scale bar 5 µm. B , D, G, H. Quantification of the 1-PLA data with CellProfiler. Significant differences between two groups were calculated with PRISM 10 Student’s t-test.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: Western blot analysis of free GEF-H1 in the lysate of Ramos and CD32b-tr cells as well as reconstituted Ramos cells expressing CD20 WT, Nmut, Cmut or NCmut after activation with RTX for 5 min. The amount of GEF-H1 purified with the GST-RhoAG17A beads and in the total lysate is shown at the top and bottom, respectively. F. Quantification of the Western blot data with Image Studio Lite (Licor) showing the difference of signal intensity in the immunoprecipitate (IP) versus that of the total lysates (TL).

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Western Blot, Expressing, Activation Assay, Purification

Journal: bioRxiv

Article Title: Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway

doi: 10.1101/2025.05.29.656301

Figure Lengend Snippet: Airyscan confocal microscopy images of Ramos CD32b-tr cells A. unstimulated or B. incubated with RTX for 20 min. Cells were fixed and stained for α-tubulin with Alexa Fluor 555 to visualize MTs. Images present the mid-section of an Airyscan processed z-stack. White arrows in B. indicate the peeling of the curved protofilaments away from MT in the treated cells. Scale bar: 5µm.

Article Snippet: The human Burkitt lymphoma B cell line Ramos was obtained from American Type Culture Collection (ATCC, Ramos cat. CRL-1923, RRID: CVCL 1646) and stably transfected with ecotropic receptor (EcoR) to allow murine retroviral transfection with MMLV particles.

Techniques: Confocal Microscopy, Incubation, Staining

Journal: Nature Communications

Article Title: TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

doi: 10.1038/s41467-024-51166-3

Figure Lengend Snippet: a – c Resting or BCR-stimulated primary human B cells from the blood of healthy donors ( a ) or Ramos B cells ( b , c ) were subjected to immunoblot analysis of total TFEB and phospho-S142 TFEB. Quantification of phospho-S142 TFEB in ( a , b ) was normalized to β-actin and is depicted as mean ± SD of n = 3 independent experiments. Statistical significance was computed using ( a ) an unpaired two-tailed Student’s t-test or ( b ) Tukey-corrected one-way ANOVA. d Schematic representation of reported TFEB phosphorylation sites (P) within individual TFEB domains indicated by GLN (glutamin-rich), AD (transcriptional activation), bHLH (basic helix-loop helix), Zip (leucine zipper), Pro, (proline-rich) and NLS (nuclear localization site). e Ramos transductants expressing TFEB variants were left untreated (−) or BCR-stimulated (+), and TFEB translocation was analyzed by imaging flow cytometry as described before. f CD19 + B cells of age-matched C57BL/6 mice were treated with DMSO, incubated with 20 nM leptomycin B (LMB) or BCR-stimulated for 60 min. g Wild-type Ramos B cells were left untreated (−) or BCR-activated (+, 60 min) in the presence of the following pharmacological agents: PP2, BAY61-3606 or ibrutinib (inhibiting Src, Syk or Btk, respectively), or the Ca 2+ chelator BAPTA-AM, or cyclosporin A (calcineurin inhbitor) or okadaic acid (inhibitor of PP1 and PP2) and analyzed for TFEB translocation. h Wild-type Ramos B cells were treated with anti-BCR antibodies or 10 µg/ml anti-CD19 antibodies as indicated. i – l Nuclear translocation of TFEB in wild-type Ramos ( i and k ) or CD19 + splenic B cells of age-matched C57BL/6 mice ( j and l ) were left untreated (−) or BCR-activated (+, 60 min) in the presence of the following pharmacological inhibitors: Wortmannin, LY294002 (both PI3K), rapamycin (mTOR), torin-1 (ATP-competitive mTOR inhibitor), PD98059 (ERK), BIO-Acetoxime (GSK3β), CHIR99021 (GSK3β) or Gö6983 (PKC). Data is depicted as mean ± SD of n = 3 ( g , h , k and l ) or n = 4 ( e , f , i and j ) independent experiments. Statistical significances were computed using Tukey-corrected one-way ANOVA. m Immunoblot analysis of TFEB phosphorylation in Ramos B cells left untreated or BCR-activated in the presence of the indicated kinase inhibitors. n Schematic representation of the examined signaling network. This graphical overview was created with BioRender.com under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. Source data are provided as a Source Data file.

Article Snippet: The human Burkitt lymphoma cell line Ramos (DSMZ ACC603), murine BALB/c-derived IIA1.6 B cells (kindly provided by Dr. Jürgen Frey) and the murine lymphoma cell line WEHI-231 (ATCC CRL-1702), derived from a BALB/c×NZB mouse, were cultured and stimulated with F(ab’) 2 fragments of anti-IgM and anti-IgG (Jackson Immuno Research).

Techniques: Western Blot, Two Tailed Test, Phospho-proteomics, Activation Assay, Expressing, Translocation Assay, Imaging, Flow Cytometry, Incubation

Journal: Nature Communications

Article Title: TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

doi: 10.1038/s41467-024-51166-3

Figure Lengend Snippet: Wild-type or three independently generated TFEB mutants of either Ramos ( a , c , and h ) or WEHI-231 cells ( b and e ) were left untreated or BCR-stimulated for the indicated time periods. Subsequently, cell surface expression of MHC class II proteins ( a , b ), the cytokine receptors CCR7 ( c ), IL-7 R ( d ) and CXCR4 ( e ) was analyzed by flow cytometry. f The migratory capability of wild-type or TFEB mutant WEHI-231 cells towards CXCL12 was measured through transwell migration assays. Data in ( a – f ) are presented as mean ± SD of n = 4 independent experiments. Statistical significances were computed using one-way ANOVA and corrected for multiple testing via Dunnett’s method. g The amount of surface CXCR4 expression measured in ( e ) was plotted against the migration efficiency towards CXCL12 depicted in ( f ). The linear relationship between these parameters was analyzed using Pearson’s correlation. Statistical significance is depicted as correlation coefficient and the corresponding two-sided p value. Linear regression is shown as solid line and the 95% CI is marked with dotted curves. h HRK relative mRNA expression was quantified by qRT-PCR using the ΔΔC T -method. Data is presented as mean of n = 3 independent experiments (with the box depicting min, max and the mean values). Source data are provided as a Source Data file.

Article Snippet: The human Burkitt lymphoma cell line Ramos (DSMZ ACC603), murine BALB/c-derived IIA1.6 B cells (kindly provided by Dr. Jürgen Frey) and the murine lymphoma cell line WEHI-231 (ATCC CRL-1702), derived from a BALB/c×NZB mouse, were cultured and stimulated with F(ab’) 2 fragments of anti-IgM and anti-IgG (Jackson Immuno Research).

Techniques: Generated, Expressing, Flow Cytometry, Mutagenesis, Migration, Quantitative RT-PCR