Journal: Frontiers in Immunology

Article Title: Tumor cells express and maintain HMGB1 in the reduced isoform to enhance CXCR4-mediated migration

doi: 10.3389/fimmu.2024.1358800

Figure Lengend Snippet: CXCL12 and HMGB1 are expressed in MCF-7, MDA-MB-231, and PC-3 cancer cell lines. (A, B) qRT-PCR analysis (normalized on 18S ) is shown for CXCL12 and HMGB1 mRNA in cancer cells. Data are shown as mean ± SEM of 5 independent experiments. Statistical analysis of the differences among the cell lines was performed using one way ANOVA, followed by Tukey’s multiple comparison test: **p < 0.01. (C) CXCL12 and HMGB1 expression assessed by confocal microscopy. In the merged images, CXCL12 is shown in red and HMGB1 in green. Nuclei and cell membranes were counterstained with DAPI (blue) and MemBrite Fix Dye (cyan), respectively. Representative images, of at least 3 independent experiments, are shown. Scale bar represents 20 µm. (D, E) CXCL12 (D) and HMGB1 (E) expression was evaluated in at least 40 cells and expressed as fluorescence intensity. Horizontal lines represent means. Statistical analysis of the differences among the cell lines was performed using one way ANOVA, followed by Tukey’s multiple comparison test: *p < 0.05, ****p < 0.0001. (F) Confocal microscope z-stack overlays showing the intracellular localization of CXCL12 in MCF-7 cells (scale bar 20 μm). CXCL12 is shown in red, while nuclei were counterstained with DAPI (blue) and cell membranes with the MemBrite Fix Dye (cyan).

Article Snippet: Nuclei were counterstained with 2 mM DAPI (62248, Thermo Fisher Scientific), and the cell membrane was stained using the MemBrite Fix cell surface staining kit from Biotium, following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Comparison, Expressing, Confocal Microscopy, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Tumor cells express and maintain HMGB1 in the reduced isoform to enhance CXCR4-mediated migration

doi: 10.3389/fimmu.2024.1358800

Figure Lengend Snippet: CXCL12 uptake by cancer cells. (A) qRT-PCR analysis (normalized on 18S ) is shown for CXCR4 mRNA expression in cancer cells. Data are shown as mean ± SEM of 5 independent experiments. (B) CXCR4 expression analyzed by flow cytometry in MCF-7, MDA-MB-231, and PC-3 cells. Data are shown as relative MFI (rMFI, mean ± SEM) of 3 independent experiments. (C) Uptake of CXCL12-Atto647 by cancer cells analyzed by flow cytometry. Left: A representative histogram showing fluorescence intensity of unstimulated (blue) and CXCL12-Atto647 stimulated cells (red). Right: CXCL12-Atto647 uptake shown as rMFI (mean ± SEM) of 5 independent experiments. (D) Uptake of CXCL12-Atto674 by cancer cells treated with the CXCR4 antagonist AMD3100. Chemokine uptake shown as rMFI (mean ± SEM) of 6 independent experiments. Statistical analysis of the differences among the cell lines was performed using two-way ANOVA, followed by Šídák’s multiple comparisons test: ****p < 0.0001. (E) CXCL12-Atto647 uptake assessed by confocal microscopy in MCF-7, MDA-MB-231, and PC-3 cells. Left: In the merged images, CXCL12 is shown in red. Nuclei and cell membranes were counterstained with DAPI (blue) and MemBrite Fix Dye (cyan), respectively (scale bars: 20 μm). Right: CXCL12-Atto647 uptake was evaluated in at least 10 cells and expressed as fluorescence intensity. Horizontal lines represent the mean values. Statistical analysis of the differences among the cell lines was performed using one way ANOVA, followed by Tukey’s multiple comparison test: *p < 0.05, ****p < 0.0001.

Article Snippet: Nuclei were counterstained with 2 mM DAPI (62248, Thermo Fisher Scientific), and the cell membrane was stained using the MemBrite Fix cell surface staining kit from Biotium, following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Confocal Microscopy, Comparison

Journal: Biochemistry and Biophysics Reports

Article Title: Soluble expression of recombinant coagulation factor IX protein using Escherichia coli

doi: 10.1016/j.bbrep.2024.101714

Figure Lengend Snippet: (A) Plasmid DNA maps for the expression of C-terminal His-tag-conjugated FIX (FIX-H) and N-terminal His-tag-conjugated FIX-coding gene (H-FIX). P, TEE, His (H), FXa, MCS, dot, and AmpR indicates primer, translation enhancing element sequence, His-tag, Factor Xa sequence, multiple cloning site, stop codon, and ampicillin resistance gene, respectively; (B) Schematic representation of the entire protein production system; T.F. indicates transformation; (C) SDS-PAGE analysis of FIX-H or H-FIX. An arrow indicates the location of target protein.

Article Snippet: HRP-conjugated anti-His-tag antibody (105,327-MM02T-H) and rabbit anti-FIX antibody (11,503-R034) were obtained from Sino Biological (Beijing, China).

Techniques: Plasmid Preparation, Expressing, Sequencing, Clone Assay, Transformation Assay, SDS Page

Journal: Biochemistry and Biophysics Reports

Article Title: Soluble expression of recombinant coagulation factor IX protein using Escherichia coli

doi: 10.1016/j.bbrep.2024.101714

Figure Lengend Snippet: (A) SDS-PAGE analysis of the pET28a-, PGK-conjugated pET28a-, or GST-conjugated pGEX4T1-coded FIX proteins; (B) SDS-PAGE analysis of rFIX proteins and empty pCold-I-coded protein (a pCold-I vector without inserting an FIX-expressing gene) before or after induction; (C) SDS-PAGE analysis of total (T) or soluble (S) fraction of rFIX proteins and empty pCold-I-coded protein after induction. Arrows indicate the location of target protein.

Article Snippet: HRP-conjugated anti-His-tag antibody (105,327-MM02T-H) and rabbit anti-FIX antibody (11,503-R034) were obtained from Sino Biological (Beijing, China).

Techniques: SDS Page, Plasmid Preparation, Expressing