Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: ICAM2 localization in rat dental pulp tissue and ICAM2 expression in HDPCs. ( A ) The mRNA expression of ICAM1 , ICAM2 (black column), ICAM3 , ICAM4 , ICAM5 in HDPC-5Y was assessed by quantitative RT-PCR (qRT-PCR). It was normalized against β-actin expression (means ± SD; n = 4; ** p < 0.01). ( B ) Hematoxylin-eosin (H&E) staining of tissue sections (sagittal sections) of mandibular first molars from Wistar rats. The right panel is the higher magnification view of boxed area in the left panel. PU: dental pulp tissue; De: Dentin. Bars, 100 μm. ( C – E ) Immunofluorescence (IF) staining of ICAM2 in the normal dental pulp tissue ( C ). The higher magnification view of boxed area in ( C , D ). Positive staining was indicated by arrow heads (odontoblasts) and arrow (dental pulp cells). Negative control: rabbit IgG (cIgG; ( E )). Nuclei were stained with DAPI (Blue). Bars, 100 μm. ( F , G ) The expression of ICAM2 in HDPC-5Y was examined by IF staining. Anti-ICAM2: Green ( F ), anti-rabbit IgG (control IgG: cIgG; ( G )). Nuclei were stained with DAPI (Blue). Bars, 100 μm. Arrow heads indicate ICAM2-positive HDPCs ( F ). ( H ) The gene expression of ICAM2 in three HDPCs (HDPC-5Y, 5L, and 5I) was examined by semi-qRT-PCR. It was normalized against GAPDH expression. ( I ) The expression intensities of ICAM2 (CD102) of HDPC-5Y (orange line) were demonstrated by flow cytometry. In the gated region, positive cells. Gray line indicates negative control (rabbit IgG).

Article Snippet: HDPCs were separated into ICAM2-positive and ICAM2-negative HDPCs by MACS using a bead-conjugated antibody against CD102 (ICAM2; Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Negative Control, Control, Gene Expression, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: ICAM2 expression in rat dental pulp tissue after direct pulp capping. ( A , B ) H&E staining of the rat dental pulp tissue after 7 days of treatment. ( B ) Higher magnification views of the boxed area in ( A ). DP: dental pulp tissue; DE: dentin; Arrowhead: reparative dentin. Bars, 100 μm. ( C – I ) IF staining of ICAM2 in the normal dental pulp tissue ( C ) and dental pulp tissue at 1 ( D ), 3 ( E ), 5 ( F ), 7 ( G ), and 14 days ( H ) post-direct pulp capping operation. Anti-ICAM2 (Green); Nuclei were stained with DAPI (Blue). Bars, 100 μm. Arrow heads indicate ICAM2-positive cells ( E ). ( I ) The number of ICAM2-positive DPCs was quantified (means ± SD; n = 3; ** p < 0.01).

Article Snippet: HDPCs were separated into ICAM2-positive and ICAM2-negative HDPCs by MACS using a bead-conjugated antibody against CD102 (ICAM2; Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: Effect of ICAM2 knockdown on odontoblast-like differentiation of HDPCs. ( A ) The expression of ICAM2 mRNA in HDPC-5Y transfected with negative control siRNA (siCont.) or ICAM2 siRNA (siICAM2) was assessed by qRT-PCR (UT, Untreated; means ± SD; n = 4; ** p < 0.01). ( B ) The expression of ICAM2 in HDPC-5Y was examined by IF staining. Anti-ICAM2: Green; Nuclei were stained with DAPI (Blue). Bars, 100 μm. ( C , D ) The formation of mineralized nodules in HDPC-5Y transfected with siCont or siICAM2 was examined by alizarin red S (ARS) staining after culture in odontoblast-like differentiation medium (DM) for 3 weeks. n = 3. The bar, 5 mm. ( D ) The graph shows quantitative analysis of the area of each ARS-positive region, which was imaged and measured using a Biozero digital microscope (means ± SD; n = 3; ** p < 0.01). ( E ) The gene expression of DSPP , Nestin , TH, and OPN in HDPC-5Y transfected with siCont or siICAM2, which were cultured with DM for 7 days, was assessed by qRT-PCR. It was normalized against β-actin expression (means ± SD; n = 4; ** p < 0.01, * p < 0.05).

Article Snippet: HDPCs were separated into ICAM2-positive and ICAM2-negative HDPCs by MACS using a bead-conjugated antibody against CD102 (ICAM2; Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Staining, Microscopy, Gene Expression, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: Effect of ICAM2-expressing HDPCs on odontoblast-like differentiation. ( A ) Schema of HDPCs separated using magnetic cell sorting (MACS) and the culture supernatants of each separated cell collected. ( B ) The gene expression of ICAM2 in ICAM2-negative HDPCs (ICAM2(−) HDPCs) and ICAM2-positive HDPCs (ICAM2(+) HDPCs) after separation using MACS was assessed by quantitative RT-PCR. It was normalized against β-actin expression (means ± SD; n = 4; ** p < 0.01). ( C ) The expression of ICAM2 in HDPC-5Y was examined by immunofluorescence staining. Anti-ICAM2: Green; Nuclei were stained with DAPI (Blue). Bars, 100 μm. ( D ) The formation of mineralized nodules in HDPC-5Y was examined by ARS staining after culture in DM with ICAM2(−) HDPCs or ICAM2(+) HDPCs supernatant for 3 weeks. n = 3. The bar, 5 mm. ( E ) The graph shows quantitative analysis of the area of each ARS-positive region, which was imaged and measured using a Biozero digital microscope (means ± SD; n = 3; ** p < 0.01). ( F , G ) The gene expression of odontoblast-related markers ( DSPP , Nestin , TH and OPN ) ( F ) and mineralization-inhibitory factors ( βig-h3 and Wnt5a ) ( G ) in ICAM2(−) HDPCs and ICAM2(+) HDPCs were assessed by quantitative RT-PCR. It was normalized against β-actin expression (means ± SD; n = 4; ** p < 0.01, * p < 0.05). ( H ) The concentration of secreted Wnt5a protein in ICAM2(−) HDPCs and ICAM2(+) HDPCs supernatant was determined by ELISA.

Article Snippet: HDPCs were separated into ICAM2-positive and ICAM2-negative HDPCs by MACS using a bead-conjugated antibody against CD102 (ICAM2; Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions.

Techniques: Expressing, FACS, Gene Expression, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: Schematic diagram suggesting the process of reparative dentin formation after direct pulp capping. By the 3 days after direct pulp capping, ICAM2-positive DPCs increased near the pulp capping area, and the mineralization-inhibitory factors secreted by these cells may have suppressed the differentiation of DPCs into odontoblast-like cells. This action may prevent excessive reparative dentin formation after direct pulp capping, restoring dental pulp tissue homeostasis. The dashed box area in the upper left figure indicates the direct pulp capping area (other five figures).

Article Snippet: HDPCs were separated into ICAM2-positive and ICAM2-negative HDPCs by MACS using a bead-conjugated antibody against CD102 (ICAM2; Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Function and Role of Intercellular Adhesion Molecule 2 in Dental Pulp Cells and Tissue

doi: 10.3390/ijms262412006

Figure Lengend Snippet: ICAM2 localization in rat dental pulp tissue and ICAM2 expression in HDPCs. ( A ) The mRNA expression of ICAM1 , ICAM2 (black column), ICAM3 , ICAM4 , ICAM5 in HDPC-5Y was assessed by quantitative RT-PCR (qRT-PCR). It was normalized against β-actin expression (means ± SD; n = 4; ** p < 0.01). ( B ) Hematoxylin-eosin (H&E) staining of tissue sections (sagittal sections) of mandibular first molars from Wistar rats. The right panel is the higher magnification view of boxed area in the left panel. PU: dental pulp tissue; De: Dentin. Bars, 100 μm. ( C – E ) Immunofluorescence (IF) staining of ICAM2 in the normal dental pulp tissue ( C ). The higher magnification view of boxed area in ( C , D ). Positive staining was indicated by arrow heads (odontoblasts) and arrow (dental pulp cells). Negative control: rabbit IgG (cIgG; ( E )). Nuclei were stained with DAPI (Blue). Bars, 100 μm. ( F , G ) The expression of ICAM2 in HDPC-5Y was examined by IF staining. Anti-ICAM2: Green ( F ), anti-rabbit IgG (control IgG: cIgG; ( G )). Nuclei were stained with DAPI (Blue). Bars, 100 μm. Arrow heads indicate ICAM2-positive HDPCs ( F ). ( H ) The gene expression of ICAM2 in three HDPCs (HDPC-5Y, 5L, and 5I) was examined by semi-qRT-PCR. It was normalized against GAPDH expression. ( I ) The expression intensities of ICAM2 (CD102) of HDPC-5Y (orange line) were demonstrated by flow cytometry. In the gated region, positive cells. Gray line indicates negative control (rabbit IgG).

Article Snippet: HDPCs were separated into ICAM2-positive and ICAM2-negative HDPCs by MACS using a bead-conjugated antibody against CD102 (ICAM2; Miltenyi Biotec, Bergisch Gladbach, Germany) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Negative Control, Control, Gene Expression, Flow Cytometry

Journal: bioRxiv

Article Title: Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein

doi: 10.1101/2024.04.12.589258

Figure Lengend Snippet: ( A-B ) Quantification of the transcript expression levels of representatives of IFN-I/λ-signaling ( i.e., MxA, ISG15 and IFNλ1 ) and NF-KB-related pathway ( i.e., TNF and IL-6 ) determined at 6 d.p.e. of HepG2/C3A ( A ) and PLC3 (B) cells, in the absence (left panels) or in co-culture with pDCs for 18 hours (right panels), as indicated. Bars represent copy number per µg total RNA; means ± SD; each dot represents one independent experiment (n=5 to 7). Statistical analysis was done using Wilcoxon rank sum test with continuity correction; p values as: * ≤0.05, ** ≤0.005 and *** ≤0.0005. (C-E) pDCs were co-cultured with HEV-electroporated PLC3 cells, their supernatants, in various settings or treated by inhibitors, as indicated, for 18 hours. (C) Quantification of IFNα in supernatants of pDCs co-cultured with HEV-infected PLC3 cells [HEV cells] or treated with supernatants from HEV infected cells [HEV SN] versus in the absence of pDCs [no pDC]; the uninfected [cont] cells were electroporated without HEV RNA and used as negative control. Bars represent means ± SD and each dot represents one independent experiment (n=4). ( D ) Quantification of IFNα in supernatants of pDCs in co-culture or separated from HEV-electroporated PLC3 cells by a permeable membrane (0.4 μm) of transwell [transwell setting]. The TLR7 agonist, imiquimod [IMQ] was used as positive control. Results presented as in C ; (n=4). ( E ) Co-culture of pDCs and HEV-electroporated PLC3 cells were treated by inhibitors of TLR7 [IRSS661], blocking antibodies against α L β 2 -integrin and ICAM-1, followed by the quantification of IFNα in supernatants of the co-cultures. Results presented as in C ; n>3 independent experiments. Statistical analysis was done using Wilcoxon rank sum test with continuity correction; p values as: * ≤0.05, ** ≤0.005 and *** ≤0.0005. (F-G) Quantification of HEV RNA replication levels in HepG2/C3A ( F ) and PLC3( G) cells, in the absence or in co-culture with pDCs, as indicated; means ± SD; each dot represents one independent experiment in terms of fold expression compared to [no pDC] condition; (n=4) for HepG2/C3A cells and PLC3 cells. Statistical analysis was done using Wilcoxon rank sum test with continuity correction and p value adjustment with Bonferroni method; p values as: * ≤0.05, ** ≤0.005 and *** ≤0.0005. (H) Fold-change in HEV-infected (ORF2+) PLC3 cells in the absence and presence of pDCs for 48 hours (n=4). (I-J) HEV-infected PLC3 cells (GFP - ORF2 + ) and uninfected cells (GFP + ORF2 - ) were co-cultured in the presence and absence of pDCs for 48 hours. (I) Results are presented as representative dot blots. ( J ) Fold-change in ORF2 + GFP - and ORF2 + GFP + cells was quantified by flow cytometry. Bars represent means ± SD and each dot per independent experiment (n=5). Statistical analysis was done using Wilcoxon rank sum test with continuity correction and p value adjustment with Bonferroni method; p values as: * ≤0.05, ** ≤0.005 and *** ≤0.0005.

Article Snippet: The following antibodies were used: mouse anti-α L integrin (clone 38; Antibodies Online); mouse anti-ICAM-1 (Clone LB-2; BD Bioscience); mouse anti-HEV ORF2 1E6, (Millipore, antibody registry #AB-827236), mouse anti-ORF2i/g/c P3H2 ( ) and mouse anti-ORF2i P1H1 ( ) .

Techniques: Expressing, Co-Culture Assay, Cell Culture, Infection, Negative Control, Membrane, Positive Control, Blocking Assay, Flow Cytometry