cd44 im7  (Bio X Cell)

Journal: Nature Communications

Article Title: Extracellular matrix mediates circulating tumor cell clustering in triple-negative breast cancer metastasis

doi: 10.1038/s41467-026-69007-w

Figure Lengend Snippet: a Maximum intensity projection of LM2 cells fixed in suspension and stained for HA (green) after 1 h of Control (Ctrl) or HAse treatment. b Representative images and quantification of numbers of clusters formed by Ctrl and HAse-treated LM2 cells after 1 h of clustering ( n = 3 (Ctrl) or 4 (HAse) biological replicates; P = 0.0020). See Supplementary Fig. for larger fields of view. c Representative image of SUM159 cells after 1 h of clustering. d Quantification of the number of clusters formed by LM2 and SUM159 cells after 1 h of clustering ( n = 3 biological replicates; P = 0.0012). e Maximum intensity projection of LM2 and SUM159 cells fixed in suspension and stained for HA (green) and CD44 (red). See Supplementary Fig. for single channel images. f Maximum intensity projection of SUM159 Ctrl and HAS2 OE cells fixed in suspension and stained for HA (green). g Representative images and quantification of numbers of clusters formed by SUM159 Ctrl and HAS2 OE cells after 1 h of clustering ( n = 3 biological replicates). h Crystal structure of the interaction between HA (green) and mouse CD44 HA binding domain (red). Dotted black lines represent hydrogen bonds. The CD44 residue corresponding to human R41 is highlighted in blue. Box indicates the position of the magnified region below. Generated using the structure deposited at Protein Data Bank ID 2JCR and the UCSF ChimeraX software. i , j Results for 293FT control and 293FT co-transfected with either HAS2 + CD44 WT or HAS2 + CD44 R41A are shown. Representative images and quantification of numbers of clusters formed by 293FT cells after 1 h of clustering ( n = 3 biological replicates; P = 0.0014, 0.9990) ( i ). Maximum intensity projection of freshly detached cells stained for HA (green) and CD44 (red) ( j ). k Quantification of the number of clusters formed by IgG- or CD44 blocking antibody IM7-treated LM2, WHIM12, and SUM159 HAS2 OE cells after 1 h of clustering ( n = 3 biological replicates; P = 0.0189, 0.0005, 0.0118). Representative images ( l ) and quantification ( m ) of the number of clusters formed by control and HAse-treated non-breast cancer cell lines ( n = 3 biological replicates; P = 0.0197, 0.0004, 0.0170). Data are represented as mean ± SEM. Statistical significance: ns = not significant; * P = < 0.05; ** P = < 0.01; *** P = < 0.001; **** P = < 0.0001 (unpaired two-sided t test ( b , d , g , k , m ) or ordinary one-way ANOVA ( i )). DAPI (blue) served as a nuclear counterstain.

Article Snippet: For immunofluorescence, the following antibodies were used: CD44-IM7 (Santa Cruz Biotechnology sc-18849; 1:100), DSG2 (Proteintech 21880; 1:100), DSC2 (ABclonal A10211; 1:100), DSP (ABclonal A7635; 1:100), Tubulin (Proteintech 11224-1-AP; 1:100), Ki67 (eBioscience 14-5698-82; 1:100), GFP (Abcam ab13970; 1:100), RFP (Rockland 600-401-379; 1:100), Ly6G-AF594 (Biolegend 127636; 1:50), Pan-cytokeratin (Santa Cruz Biotechnology sc-8018; 1:100), Cytokeratin 19 (Invitrogen MA5-12663; 1:100), EpCAM-AF488 (Cell Signaling 5198S; 1:50), and EGFR-AF488 (Biolegend 352908; 1:50).

Techniques: Suspension, Staining, Control, Binding Assay, Residue, Generated, Software, Transfection, Blocking Assay

Journal: Nature Communications

Article Title: Extracellular matrix mediates circulating tumor cell clustering in triple-negative breast cancer metastasis

doi: 10.1038/s41467-026-69007-w

Figure Lengend Snippet: a 3D projection of a freshly detached LM2 cell fixed in suspension. Box indicates the position of the 3-fold magnified region on the right which shows the localization of HA at the tips of protrusions. Cells were labeled for HA (green) and CD44 (red). b Quantification of longest protrusion length in LM2 Ctrl, HAse-treated and CD44KO cells ( n = 3 biological replicates; P = 0.4640, 0.7294). Protrusion length was quantified from actin-labelled protrusions. c Maximum intensity projection of fixed early-interacting LM2 cells stained for HA (green) and CD44 (red). Box indicates the position of the 2-fold magnified region on the right demonstrating HA localization at sites of interacting protrusions. d Left: single optical section of a detached LM2 cell stained for CD44 (green) and actin (red). Box indicates the position of the 3-fold magnified region on the right, showing protrusions. Line indicates position of the line profile (right) for CD44 (green) and actin (red) signals in protrusion. See Supplementary Fig. for tubulin staining of the same cell. e Quantification of the number of clusters formed by LM2 cells treated with Latrunculin A (LA) or Colchicine (Col) after 1 h of clustering ( n = 3 biological replicates; P = 0.0052, 0.3744). See Supplementary Fig. for number of remaining single cells. f Live imaging of early interaction events between freshly detached LM2 cells. Single optical sections are shown; cellular protrusions were visualized using anti-CD44 staining. Minutes that passed since the start of the experiment are displayed above each picture. See Supplementary Movies – and Supplementary Fig. for another example of the maturation process. g Live imaging of freshly detached LM2 Ctrl and LM2 CD44KO cells. Cells were labeled for CD44 (green) and actin (red). Arrows indicate interactions between Ctrl cells (cyan) or KO cells (yellow). Minutes that passed since the start of the experiment are displayed above each picture. See Supplementary Movie . h Quantification of live imaging experiment shown in ( g ). Distance between Ctrl cells (cyan) or KO cells (yellow) over time is displayed ( n = 3 biological replicates). i Transmission electron microscopy pictures depicting the five types of interactions observed in LM2 WT cells after clustering for 5–20 min. Images were taken at 5000–15000x magnification. Purple and yellow dotted lines indicate membrane regions involved in each interaction. See also Supplementary Fig. . j Number of connections between cells exemplified in ( i ). Results were categorized by distance between interacting cells ( n = 3 biological replicates). k Quantification of the cell-cell interaction types depicted in ( i ) Interactions were categorized by distance between interacting cells. Data are represented as mean ± SEM. Statistical significance: ns = not significant; ** P = < 0.01 (ordinary one-way ANOVA). DAPI (blue) served as a nuclear counterstain.

Article Snippet: For immunofluorescence, the following antibodies were used: CD44-IM7 (Santa Cruz Biotechnology sc-18849; 1:100), DSG2 (Proteintech 21880; 1:100), DSC2 (ABclonal A10211; 1:100), DSP (ABclonal A7635; 1:100), Tubulin (Proteintech 11224-1-AP; 1:100), Ki67 (eBioscience 14-5698-82; 1:100), GFP (Abcam ab13970; 1:100), RFP (Rockland 600-401-379; 1:100), Ly6G-AF594 (Biolegend 127636; 1:50), Pan-cytokeratin (Santa Cruz Biotechnology sc-8018; 1:100), Cytokeratin 19 (Invitrogen MA5-12663; 1:100), EpCAM-AF488 (Cell Signaling 5198S; 1:50), and EGFR-AF488 (Biolegend 352908; 1:50).

Techniques: Suspension, Labeling, Staining, Imaging, Transmission Assay, Electron Microscopy, Membrane

Journal: Nature Communications

Article Title: Extracellular matrix mediates circulating tumor cell clustering in triple-negative breast cancer metastasis

doi: 10.1038/s41467-026-69007-w

Figure Lengend Snippet: a Schematic depicting mammary fat pad injection of GFP-tagged LM2 control ( n = 5 mice) or HAS2 KD ( n = 7 mice) single cells in immunocompromised mice. These mouse samples were analyzed in experiments ( b – j ). Representative images ( b ) and quantification ( c ; P = 0.0049) of H&E-stained lungs showing metastatic tumor burden at experimental endpoint. d Average primary tumor volume for these mice over 58 days ( P = 0.2785). e Primary tumor weight at experimental endpoint ( P = 0.3963). See Supplementary Fig. for tumor pictures. f Schematic demonstrating the blood processing steps for CTC analysis. Quantification of the number of total CTCs ( g ; P = 0.0048) or CTC clusters ( h ; P = 0.0455) detected per mL of blood. See Supplementary Fig. for number of single CTCs. i , Dot plots showing the unique cluster sizes observed in each mouse in control (left) and HAS2 KD (right) groups. A value of zero indicates no CTC clusters were detected. Numbers inside the graph indicate total detected clusters. j Maximum intensity projection of a fixed LM2 CTC cluster stained for HA (green pseudo color) and CD44 (red). See Supplementary Fig. for CTC staining. k Left: schematic depicting tail vein injection of GFP-tagged LM2 control or HAS2 KD cell aggregates in immunocompromised mice ( n = 7 mice per group). These mouse samples were analyzed in experiments ( l – r ). Right: representative images of lung tumor burden measured via bioluminescent imaging in these mice at time of injection (Day 0) and upon experimental endpoint at Week 4. l Quantification of lung metastasis signal at time of injection (Day 0; P = 0.5176) and Weeks 1 ( P = 0.0673), 2 ( P = 0.0229), and 4 ( P = 0.0016). Data is normalized to lung signal at time of injection. See Supplementary Fig. for a growth curve. m , Representative images of H&E-stained lungs showing tumor burden at Week 4. Quantification of the number of total CTCs ( n ; P = 0.0064) or CTC clusters ( o ; P = 0.0047) detected per mL of blood. See Supplementary Fig. for percent of mice with CTCs and number of single CTCs. p Dot plots showing the unique cluster sizes observed in each mouse in control (left) and HAS2 KD (right) groups. A value of zero indicates no CTC clusters were detected. Numbers inside the graph indicate total detected clusters. q Single optical section of an early connection site in a LM2 CTC cluster stained for HA (green pseudo color) and CD44 (red). Box indicates the position of the 3-fold magnified region on the right which illustrates the HA-mediated connection of protrusions (outlined with dotted white lines) from adjacent cells. See Supplementary Fig. for CTC staining and Supplementary Movie for a 3D depiction of the whole interaction site. See Supplementary Fig. for an alternative z-panel. r Maximum intensity projection of a large LM2 CTC cluster stained for HA (green pseudo color) and CD44 (red). White arrowheads indicate examples of HA enrichment at cell-cell interaction sites. See Supplementary Fig. for CTC staining. Data are represented as mean ± SEM. Statistical significance: ns = not significant; * P = < 0.05; ** P = < 0.01 (unpaired two-sided t test ( c –e , l ) or two-sided Mann-Whitney test ( g –h , n–o )). Blood from 3 different Ctrl mice was analyzed to obtain the representative microscopy pictures shown in ( j , q , r ). DAPI (blue) served as a nuclear counterstain. Note: for visualization purposes, HA staining was assigned a green pseudo-color to match the color scheme used throughout the manuscript. a , f , k were created in BioRender. Bobkov, G. (2026) https://BioRender.com/srzuwrk .

Article Snippet: For immunofluorescence, the following antibodies were used: CD44-IM7 (Santa Cruz Biotechnology sc-18849; 1:100), DSG2 (Proteintech 21880; 1:100), DSC2 (ABclonal A10211; 1:100), DSP (ABclonal A7635; 1:100), Tubulin (Proteintech 11224-1-AP; 1:100), Ki67 (eBioscience 14-5698-82; 1:100), GFP (Abcam ab13970; 1:100), RFP (Rockland 600-401-379; 1:100), Ly6G-AF594 (Biolegend 127636; 1:50), Pan-cytokeratin (Santa Cruz Biotechnology sc-8018; 1:100), Cytokeratin 19 (Invitrogen MA5-12663; 1:100), EpCAM-AF488 (Cell Signaling 5198S; 1:50), and EGFR-AF488 (Biolegend 352908; 1:50).

Techniques: Injection, Control, Staining, Imaging, MANN-WHITNEY, Microscopy

Journal: Nature Communications

Article Title: Extracellular matrix mediates circulating tumor cell clustering in triple-negative breast cancer metastasis

doi: 10.1038/s41467-026-69007-w

Figure Lengend Snippet: a Left: schematic of LM2 Ctrl-alone, HAS2 KD-alone, or a 1:1 mixture of Ctrl (red) and HAS2 KD (green) single cells set up for clustering assay. Right: representative images and quantification of cluster numbers after 1 h of clustering ( n = 3 biological replicates; P = 0.0225, 0.9958). b Left: schematic of LM2 Ctrl-alone, CD44 KO-alone, or 1:1 mixture of Ctrl (red) and CD44 KO (green) single cells set up for clustering assay. Right: representative images and quantification of cluster numbers after 1 h of clustering ( n = 3 biological replicates; P = 0.0120, 0.0778) c Maximum intensity projection of fixed early-interacting LM2 control (top) and HAS2 KD (bottom) cells stained for HA (green pseudo color) and CD44 (red). Box indicates position of the 3-fold magnified region on the right demonstrating HA localization at interaction sites. See Supplementary Fig. for staining of the HAS2 KD cell. d Maximum intensity projection of an in vitro LM2 cell cluster containing GFP-tagged HAS2 KD cells (green) and RFP-tagged Ctrl cells (red) stained for HA (yellow). White arrowheads indicate HA enrichment at the cell-cell interaction site between control and HAS2 KD cells. See Supplementary Fig. for HA quantification. e Schematic depicting tail vein injection of LM2 cell aggregates in immunocompromised mice. Mixed aggregates of RFP-labeled Ctrl and GFP-labeled HAS2 KD cells were injected. f Quantification of the total number of CTCs originating from Ctrl and HAS2 KD cells per mouse. Each line represents one mouse ( n = 7 mice; P = 0.0156). Two mice had overlapping values. g Pie chart showing average proportions of CTC clusters composed of only Ctrl cells, only HAS2 KD cells, or a mixture of both Ctrl and HAS2 KD cells ( n = 3 mice). Mice with no CTC clusters were excluded from this analysis. h Quantification of the number of CTCs originating from Ctrl and HAS2 KD cells in each mixed CTC cluster. Each dot represents the number of Ctrl or HAS2 KD CTCs found in a mixed CTC cluster, with the connecting line indicating paired measurements ( n = 108 total clusters from 3 mice; many clusters had identical values). i Maximum intensity projection of a fixed LM2 CTC cluster containing RFP-tagged Ctrl cells (red) and GFP-tagged HAS2 KD cells (green) stained for HA (yellow). White arrowhead indicates HA enrichment at the cell-cell interaction site between control and HAS2 KD CTCs. j Maximum intensity projection of a TNBC patient CTC cluster consisting of two CTCs with heterogeneous HA (green) and CTC marker (red) staining. White arrowhead indicates HA enrichment at CTC-CTC interaction site. k Schematic of HA localization in early interactions and mature clusters between CTCs and non-CTCs. l Maximum intensity projection of a freshly established heterotypic LM2 CTC and non-CTC cluster stained for HA (green) and CD44 (red pseudo color). Box indicates the position of the 3-fold magnified region on the right which illustrates the HA-mediated connection of protrusions (outlined with dotted white lines) from adjacent cells. See Supplementary Fig. for CTC staining. m Maximum intensity projection of a fixed heterotypic LM2 CTC cluster stained for HA (green) and Ly6G (red pseudo color). “neu” denotes Ly6G+ neutrophil. White arrowhead indicates HA enrichment at the CTC-neutrophil interaction site. See Supplementary Fig. for CTC staining. n Maximum intensity projection of a patient CTC cluster consisting of two CTCs and a non-tumor cell. Cells were stained for HA (green) and CTC markers (red). White arrowheads indicate HA enrichment at CTC and non-CTC interaction site. Data are represented as mean ± SEM. Statistical significance: ns = not significant; * P = < 0.05; ** P = < 0.01; **** P = < 0.0001 (ordinary one-way ANOVA ( a , b ) or paired two-sided Wilcoxon test ( f )). Blood from three different mice and five different patients was analyzed to obtain the representative microscopy pictures shown in i –j, l–n . DAPI (blue) served as a nuclear counterstain. Note: for visualization purposes, green/red pseudo-color were assigned for HA/CD44/Ly6G to match the color scheme used throughout the manuscript. e , k were created in BioRender. Bobkov, G. (2026) https://BioRender.com/srzuwrk .

Article Snippet: For immunofluorescence, the following antibodies were used: CD44-IM7 (Santa Cruz Biotechnology sc-18849; 1:100), DSG2 (Proteintech 21880; 1:100), DSC2 (ABclonal A10211; 1:100), DSP (ABclonal A7635; 1:100), Tubulin (Proteintech 11224-1-AP; 1:100), Ki67 (eBioscience 14-5698-82; 1:100), GFP (Abcam ab13970; 1:100), RFP (Rockland 600-401-379; 1:100), Ly6G-AF594 (Biolegend 127636; 1:50), Pan-cytokeratin (Santa Cruz Biotechnology sc-8018; 1:100), Cytokeratin 19 (Invitrogen MA5-12663; 1:100), EpCAM-AF488 (Cell Signaling 5198S; 1:50), and EGFR-AF488 (Biolegend 352908; 1:50).

Techniques: Control, Staining, In Vitro, Injection, Labeling, Marker, Microscopy

Journal: Scientific Reports

Article Title: A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

doi: 10.1038/s41598-025-16007-3

Figure Lengend Snippet: ( A – C ) Representative CLSM images showing CD44 expression (green) in THP-1 cells, M0 macrophages, and M1 macrophages, nuclei were counter stained with DAPI (blue). Scale bar: 20 μm; ( D ) Quantitative analysis of CD44 expression in THP-1 cells, M0 macrophages and M1 macrophages using image J software (One way ANOVA, * p < 0.05).

Article Snippet: Primary antibodies such as Cadherin–11 (sc-365867), CD44 (NBP1-41266), Fibroblast specific protein (NBP1-89402), CD68 ( ABM40050 ) and HIF–1α (3716 S) were purchased from Santa Cruz Biotechnology Inc., USA, Novus Biologicals, USA, Abbkine, USA and Cell signalling Technologies, USA, respectively.

Techniques: Expressing, Staining, Software

Journal: Scientific Reports

Article Title: A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

doi: 10.1038/s41598-025-16007-3

Figure Lengend Snippet: ( A , B ) Gene expression levels of inflammatory cytokines TNF–α and IL–1β respectively, in THP-1, M0 and M1 macrophages, analyzed by qRT–PCR; ( C , D ) Quantification of TNF–α and IL–1β protein expression in resting and stimulated cells using ELISA; ( E – G ) Gene expression analysis of matrix metalloproteinase MMP2, MMP3, and MMP9 in THP-1, M0 and M1 macrophages; ( H ) Gene expression of CD44 in THP-1, M0 and M1 macrophage cells. ( n = 3; One way ANOVA, * p < 0.05).

Article Snippet: Primary antibodies such as Cadherin–11 (sc-365867), CD44 (NBP1-41266), Fibroblast specific protein (NBP1-89402), CD68 ( ABM40050 ) and HIF–1α (3716 S) were purchased from Santa Cruz Biotechnology Inc., USA, Novus Biologicals, USA, Abbkine, USA and Cell signalling Technologies, USA, respectively.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

doi: 10.1038/s41598-025-16007-3

Figure Lengend Snippet: ( A , B ) Representative SEM images of resting and stimulated synovial fibroblast-like cells from rat, respectively; ( C – E ) CLSM images of showing immunofluorescence staining of fibroblast specific protein (FSP), cadherin–11 (Cad–11) and CD44 of non–stimulated rFLS (Scale bar: 20 μm); ( F – H ) CLSM images of fibroblast specific protein (FSP), cadherin–11 (Cad–11) and CD44 of stimulated rFLS (Scale bar: 20 μm); ( I ) Quantification of the aspect ratio of non–stimulated and stimulated rFLS, indicating morphological polarization; ( J – L ) Quantitative fluorescence intensity analysis of fibroblast specific protein, cadherin-11 and CD44 in non–stimulated and stimulated rFLS (Student’s t–test, * p < 0.05).

Article Snippet: Primary antibodies such as Cadherin–11 (sc-365867), CD44 (NBP1-41266), Fibroblast specific protein (NBP1-89402), CD68 ( ABM40050 ) and HIF–1α (3716 S) were purchased from Santa Cruz Biotechnology Inc., USA, Novus Biologicals, USA, Abbkine, USA and Cell signalling Technologies, USA, respectively.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Scientific Reports

Article Title: A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

doi: 10.1038/s41598-025-16007-3

Figure Lengend Snippet: Gene and protein expression profiles of non–stimulated and cytokine-stimulated rFLS cells. ( A , B ) Gene expression of synovial fibroblast markers Thy–1 and cadherin–11; ( C – E ) Gene expression of proinflammatory cytokines TNF–α, IL–1β and IL–6; ( F – H ) ELISA-based quantification of TNF–α, IL–1β and IL–6 secretion in non-stimulated vs. stimulated rFLS cells and ( I ) Cytokine concentrations in the macrophage-derived conditioned media used for FLS stimulation; ( J – L ) Gene expression of CD44, MMP2 and MMP9, in non-stimulated vs. stimulated rFLS cells ( n = 3; Student’s t–test, * p < 0.05).

Article Snippet: Primary antibodies such as Cadherin–11 (sc-365867), CD44 (NBP1-41266), Fibroblast specific protein (NBP1-89402), CD68 ( ABM40050 ) and HIF–1α (3716 S) were purchased from Santa Cruz Biotechnology Inc., USA, Novus Biologicals, USA, Abbkine, USA and Cell signalling Technologies, USA, respectively.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Scientific Reports

Article Title: A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

doi: 10.1038/s41598-025-16007-3

Figure Lengend Snippet: ( A , B ) Representative SEM images of non-stimulated and stimulated SW982 cells; ( C – E ) Representative CLSM images showing immunofluorescence staining for fibroblast specific protein (FSP: green), cadherin–11 (Cad–11: green) and CD44 (green) in non-stimulated SW982 cells ( F – H ) Corresponding images for stimulated SW982 cells. All cells were counterstained with DAPI for nuclei visualization (blue) (Scale bar: 20 μm); ( I ) Quantification of the aspect ratio of non-stimulated and stimulated SW982 cells; ( J – L ) Quantitative fluorescence intensity analysis of FSP, Cad-11 and CD44 in non-stimulated and stimulated SW982 cells ( n = 3; Student’s t–test, * p < 0.05).

Article Snippet: Primary antibodies such as Cadherin–11 (sc-365867), CD44 (NBP1-41266), Fibroblast specific protein (NBP1-89402), CD68 ( ABM40050 ) and HIF–1α (3716 S) were purchased from Santa Cruz Biotechnology Inc., USA, Novus Biologicals, USA, Abbkine, USA and Cell signalling Technologies, USA, respectively.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Scientific Reports

Article Title: A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

doi: 10.1038/s41598-025-16007-3

Figure Lengend Snippet: Gene and protein expression analysis of non–stimulated and stimulated SW982 cells. qRT–PCR analysis of ( A ) cadherin–11, ( B ) CD44, ( C ) TNF–α, ( D ) IL–1β. ELISA quantification of inflammatory cytokine secretion levels of ( E ) TNF–α and ( F ) IL–1β. qRT–PCR analysis of matrix metalloproteinases ( G ) MMP2, ( H ) MMP3, ( I ) MMP 7 and ( J ) MMP 9. ( n = 3; Student’s t–test, * p < 0.05).

Article Snippet: Primary antibodies such as Cadherin–11 (sc-365867), CD44 (NBP1-41266), Fibroblast specific protein (NBP1-89402), CD68 ( ABM40050 ) and HIF–1α (3716 S) were purchased from Santa Cruz Biotechnology Inc., USA, Novus Biologicals, USA, Abbkine, USA and Cell signalling Technologies, USA, respectively.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay