Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: Fluorescent micrographs of HEK293 cells transfected with (A, B) SAM-6 μHC and SAM-6 λLC construct pair, (C, D) SAM-6 μHC subunit alone, or (E, F) SAM-6 λLC subunit alone. On day-2 post-transfection, transfected cells were seeded onto poly-D-lysine coated coverslips in the absence (A, C, E) or presence (B, D, F) of 15 μg/mL Brefeldin A (BFA) and cultured statically for 24 hr. On day-3 post-transfection, cells were fixed, permeabilized, and immunostained. (A, B) Co-staining was performed using FITC-labeled anti-human μHC antibody and Texas Red-labeled anti-human λLC antibody. (C, D) Co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. (E, F) Co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to make ‘overlay’ views in A and B. DIC and red image fields were superimposed to create ‘overlay’ views in C‒F.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Transfection, Construct, Cell Culture, Staining, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A) Schematic representation of SAM-6 λLC subunit (top), SAM-6 μHC subunit (middle), and SAM-6 μHC-ΔCH1 mutant which lacks the CH1 domain (bottom). The deleted CH1 domain is shown as a dotted line. Individual domain names are indicated in each box. ER targeting is driven by a heterologous signal sequence adapted from a VK1 encoding gene. (B) Fluorescent micrographs of HEK293 cells transfected with μHC (first row) or μHC-ΔCH1 mutant (second and third rows). On day-3 post-transfection, cells were fixed, permeabilized, and immunostained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views. (C) Fluorescent micrographs of HEK293 cells transfected with [λLC + μHC] pair (first row) or [λLC + μHC-ΔCH1] pair (second and third rows). Immunostaining was performed using FITC-labeled anti-human μHC antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to create ‘overlay’ views. (D‒G) HEK293 cells were transfected with μHC (lanes 1 and 5), μHC-ΔCH1 (lanes 2 and 6), [λLC + μHC] pair (lanes 3 and 7), or [λLC + μHC-ΔCH1] pair (lanes 4 and 8). Cell culture media were harvested at day-7 post-transfection and analyzed by SDS-PAGE under reducing conditions (D, E; lanes 1‒4) or non-reducing conditions (F, G; lanes 1‒4). Cell lysate samples were also prepared on day-7 post-transfection and analyzed by SDS-PAGE (D, lanes 5‒8) or Western blotting (E, lanes 5‒8) after resolving the proteins under reducing conditions. Western blotting was performed using polyclonal anti-IgM (H+L) to detect both μHC and λLC subunits simultaneously as well as assembly intermediates composed of μHC or λLC or both. A faintly detectable μHC-ΔCH1 is pointed by a black arrowhead (E, lane 4). Likewise, faintly detectable μHC-ΔCH1 covalent dimers are pointed by black arrowhead (G, lane 4). The assembled hexameric IgM product is pointed by a red arrowhead (F, G; lane 3). Identifiable assembly intermediates are labeled next to the corresponding bands in panels F and G.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Sequencing, Transfection, Labeling, Immunostaining, Cell Culture, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A, top) Schematic representation of the full-length SAM-6 λLC (top row) and its ΔCS mutant (second row) in which two C-terminal amino acids (Cys-213 and Ser-214) are deleted. (A, bottom) The position of Cys-213 residue involved in the HC‒LC inter-chain disulfide bond is highlighted in yellow in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (B, C) HEK293 cells were transfected with full-length λLC (lanes 1, 3, 5) or its ΔCS mutant (lanes 2, 4, 6). At day-7 post-transfection, cell lysates (lanes 1 and 2) and cell culture media samples (lanes 3 and 4) were prepared and resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (B) or by Western blotting (C). The day-7 cell culture media were also analyzed by Coomassie staining or Western blotting after resolving the proteins under non-reducing conditions (B and C, lanes 5 and 6). Blotted membranes were probed with polyclonal anti-λLC antibodies. The corresponding protein band for the λLC subunit is pointed by an arrowhead and labeled (lanes 1‒4). Monomeric and dimeric free λLC subunit is labeled next to lane 6. (D, E) Fluorescent micrographs of HEK293 cells transfected with full-length λLC (D) or ΔCS mutant (E). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Residue, Transfection, Cell Culture, SDS Page, Staining, Western Blot, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A, left) Schematic representation of parental SAM-6 μHC (top row) and its C137S and C337S mutants (second and third rows). (A, right) The position of Cys-137 and Cys-337 residues is highlighted in yellow in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (B, C) HEK293 cells were transfected with parental μHC and its mutants, as shown at the top of each lane. At day-7 post-transfection, cell culture media (B) and cell lysates (C) were prepared and resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (B, C, left panels) or by Western blotting (B, C, right panels). Blotted membranes in B and C were probed with polyclonal anti-IgM (H+L) antibodies. Both parental and mutant μHCs failed to secrete to the culture media. (D) Fluorescent micrographs of HEK293 cells transfected with parental μHC (top row), μHC (C137S) mutant (second and third rows), or μHC (C337S) mutant (fourth and fifth rows). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Transfection, Cell Culture, SDS Page, Staining, Western Blot, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A) Schematic representation of parental SAM-6 μHC (top row) and its 4×C>S mutant (second row). The positions of key cysteine residues important for the inter-chain disulfide formation are marked on the parental μHC. (B) The position of all four Cys residues involved in inter-chain disulfide bond formation on μHC and Cys-213 on λLC are depicted in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (C, D) HEK293 cells were transfected with parental μHC alone (lane 1) or its 4×C>S mutant alone (lane 2). Likewise, the cells are co-transfected with μHC and JC (lane 3) or μHC (4×C>S) and JC (lane 4). At day-7 post-transfection, cell lysates (C) and culture media (D) were resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (C, D; left panel) or by Western blotting (C, D; right panels). Blotted membranes in C and D were probed with polyclonal anti-IgM (H+L) antibody (top panel) or monoclonal anti-JC antibody (bottom panel). (E) Day-7 culture media were also analyzed by Western blotting after proteins were resolved under non-reducing conditions. Blotted membranes were probed with polyclonal anti-IgM (H+L) antibody (left panel) or monoclonal anti-JC antibody (right panel). (F) Fluorescent micrographs of HEK293 cells transfected with parental μHC (top row) or μHC (4×C>S) mutant (second and third rows). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views. (G) Fluorescent micrographs of HEK293 cells co-transfected with μHC and JC (top row) or μHC (4×C>S) mutant and JC (second and third rows). Cells were co-stained FITC-labeled anti-human μHC (shown in green) and monoclonal anti-JC antibody followed by AlexaFluor594-conjugated secondary antibody (shown in red). Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Transfection, SDS Page, Staining, Western Blot, Labeling

Journal: OncoImmunology

Article Title: An epitope-specific novel anti-EMMPRIN polyclonal antibody inhibits tumor progression

doi: 10.1080/2162402x.2015.1078056

Figure Lengend Snippet: Figure 1: EMMPRIN structure and peptide design, and specificity of 161-Ab: (A) Partial

Article Snippet: One strip was probed with the 1:1,000 diluted commercial rat anti-mouse EMMPRIN (MAB772, R&D systems) and then with the 1:5,000 diluted HRP-conjugated goat anti-rat IgG (112-035- D ow nl oa de d by [ U ni ve rs ity o f R eg in a] a t 0 6: 58 2 4 Fe br ua ry 2 01 6 062, Jackson) and served as positive control (P.C).

Techniques:

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 1. Validation of the CT26-KD cells. The parental CT26 cells (WT) and knocked-down CT26 cells (KD) were seeded (8 × 104 cells each) in 24-well plates in 400 µL full medium for 48 h. At the end of the incubation, (A) total RNA was extracted from the cells and amplified using EMMPRIN specific primers (n = 4), and (B) the supernatants were collected for an ELISA analysis of EMMPRIN secretion (n = 9). Data are presented as means ± SE and analyzed using the two-tailed Student’s t test analysis. (C) CT26-WT and CT26-KD cells (30,000 cells/well/300 µL) were stained as described in the methods. A representative image, demonstrating reduced EMMPRIN protein expression in the CT26-KD cells (n = 3). Bar size is 20 µM. (D) EMMPRIN is known to appear in several bands, reflecting its low and high glycosylation patterns. Western blot analysis demonstrates that in the CT26-KD cells, all EMMPRIN bands showed a reduced expression of EMMPRIN.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Biomarker Discovery, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Expressing, Glycoproteomics, Western Blot

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 2. Co-culturing enhances the secretion of EMT-driver cytokines. CT26-WT or CT26-KD cells (80,000 cells each) were each cultured alone or co-cultured with RAW 264.7 cells that were seeded in the upper chamber of the inserts (0.4 µm pore size) at a ratio of 1:1, in serum-starvation medium (final volume 650 µL) for 48 h. At the end of the incubation, supernatants were collected and the concentrations of (A) TGFβ (n = 5), (B) soluble EMMPRIN (n = 6), (C) TNFα (n = 6), and (D) IL-6 (n = 6) were determined by ELISA. Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Cell Culture, Pore Size, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 4. Expression of the EMT-TFs and dormancy markers is enhanced in the CT26-KD cells, but reduced in the co-culture or its simulation. CT26-WT or CT26-KD cells (8 × 104 cells each) were incubated alone or in co-culture with RAW 264.7 cells as described before for 48 h. Alternatively, single cultures of CT26-WT or CT26-KD cells (8 × 104 cells) were cultured with or without the addition of recombinant TGFβ (10 ng/mL) or recombinant EMMPRIN (25 ng/mL or 250 ng/mL). Total RNA was extracted from the CT26 cells, cDNA was prepared, and the genes for the dormancy markers NR2F1 and p21 or the EMT-TFs Slug and Zeb1 were amplified by qPCR as described in the methods. (A–D) cells incubated in co-cultures (n = 5–6), (E–H) single cultures incubated with the addition of TGFβ (10 ng/mL) (n = 5–6), and (I–L) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL) (n = 5). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Expressing, Co-Culture Assay, Incubation, Cell Culture, Recombinant

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 5. CT26-KD exhibits reduced proliferation. CT26-WT or CT26-KD cells (8 × 104 cells) were incubated under the same conditions as described in Figure 4. Cell proliferation was measured using (A,D,G) the CCK8 kit (n = 8) or (H) the BrdU kit (n = 7) as well as the expression of (B,E,I) the cyclin D1 mRNA (n = 5) or (C,F), and the Ki67 mRNA (n = 4). (A–C) Cells incubated in co-cultures, (D,F) single cultures incubated with the addition of TGFβ (10 ng/mL), and (G–I) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test. ***, p < 0.001 relative to the CT26-WT without addition of rec. EMMPRIN; $$$, p < 0.001 relative to CT26-WT with 25 ng/ml rec. EMMPRIN; &&&, p < 0.001 relative to the CT26-WT at each concentration.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Incubation, Expressing, Recombinant, Concentration Assay

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 6. CT26-KD exhibits reduced angiogenic potential, and the co-culture reverses it. The mouse endothelial cell line bEND3 (4 × 104 cells) was cultured in full medium in 96-well plates to confluency for 24 h. A scratch was made across the monolayer, detached cells were washed away, and the remaining bEND3 cells were incubated with conditioned media (CM) derived from previous experiments for 24 h, in order to allow the migration of cells to close the gap. The CM was diluted 1:2 with full medium, to a final volume of 100 µL. Images were taken before the addition of the CM (0 h) and after 24 h of incubation with the CM (24 h). (A) Representative images of the wound assay of co-cultures. Bar size is 250 µM. (B) Quantitation of the migration of bEND3 cells cultured with CM from co-culture experiments (n = 12), with (C) concentrations of VEGF (n = 7) and (D) MMP-9 (n = 7) in the supernatants derived from co-culture experiments. (E) Quantitation of the migration using CM derived from the TGFβ experiments (n = 5), and concentrations of (F) VEGF (n = 8) and (G) MMP-9 (n = 5) in the supernatants derived from TGFβ experiments. (H) Quantitation of the migration using CM derived from the recombinant EMMPRIN experiments (n = 9), and concentrations of (I) VEGF (n = 9) and (J) MMP-9 (n = 7) in the supernatants derived from recombinant EMMPRIN experiments. Data are presented as means ± SE, and analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Co-Culture Assay, Cell Culture, Incubation, Derivative Assay, Migration, Quantitation Assay, Recombinant

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 7. The combination of recombinant TGFβ and EMMPRIN has no effect on the addition of TGFβ or EMMPRIN alone. CT26-WT or CT26-KD cells (2.5 × 104 cells) were incubated in triplicates for 48 h in serum starvation medium, with or without the addition of recombinant TGFβ (5 ng/mL), EMMPRIN (5 ng/mL), or their combination. The effect on the (A) proliferation, as measured by CCK8 (n = 10), (B) the concentrations of secreted VEGF (n = 5), as well as the mRNA expression of (C) NR2F1 (n = 6), (D) p21 (n = 6), (E) Slug (n = 6), and (F) Zeb1 (n = 6). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Recombinant, Incubation, Expressing

Journal: Frontiers in Microbiology

Article Title: Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection

doi: 10.3389/fmicb.2019.01181

Figure Lengend Snippet: Characterization of Nm interaction with iPSC-BECs. (A) Schematic cartoon of Nm strains used. MC58 is a serogroup B strain, MC58Δ siaD is an isogenic non-capsulated mutant, 8013/12 is a serogroup C strain and 8013/12Δ pilT is a highly piliated mutant. (B) Gentamicin protection assay of Nm on iPSC-BECs showing invasion of MC58Δ siaD and 8013/12 relative to MC58 into iPSC-BECs at the indicated time points and Multiplicity of Infection (MOI) of 10. (C) Confocal microscopy images of iPSC-BECs infected with the Nm strains mentioned in (B) , at 4 h p.i. and MOI 100. Image is a maximum image projection. Scale bar = 20 μm. (D) Gentamicin protection assay showing invasion of Δ pilT mutant relative to WT 8013/12 Nm strains into iPSC-BECs at 4 h p.i. and MOI 10. For (B,D) data is presented as mean ± S.E.M of three independent experiments done in technical duplicate and triplicate, respectively. Student’s t -test was used to determine significance. ∗ p < 0.05; ∗∗ p < 0.01. (E) Immunofluorescence staining showing areas of recruitment of receptor CD147 (red) around MC58 and MC58Δ siaD colonies (green) highlighted with white arrow heads in iPSC-BECs at 4 h p.i. and MOI of 100. Scale bar = 5 μm.

Article Snippet: Primary antibodies [ZO-1 (1:100, Proteintech, #21773-1-AP), Claudin-5 (1:100, Abcam, #ab15106), Occludin (1:200, Thermo, #33-1500), and CD147 (1:100, Biorad, #MCA2882Z)] were incubated overnight at 4°C.

Techniques: Mutagenesis, Infection, Confocal Microscopy, Immunofluorescence, Staining