Journal: International Journal of Molecular Sciences

Article Title: The Role of C-X-C Chemokine Receptor Type 4 (CXCR4) in Cell Adherence and Spheroid Formation of Human Ewing’s Sarcoma Cells under Simulated Microgravity

doi: 10.3390/ijms20236073

Figure Lengend Snippet: ( a ) EWS/FLI1 expression was significantly upregulated in both spheroids and adherent cells compared to the 1 g control group (18.5x, 8.2x, p < 0.05 each). ( b ) CXCR4 was significantly upregulated in spheroids compared to control and adherent cells (27x, 30x, * p < 0.05 each). ( c ) DKK2 was downregulated in spheroids (0.8x, p < 0.05). ( d ) VEGF-A expression significantly decreased in adherent cells under s-µg (0.73x, * p < 0.05). ( e ) CD44 was significantly upregulated in spheroids after 24 h (3.7x, * p < 0.05). ( f ) CAV1 was significantly upregulated in spheroids and in adherent cells (1.3x, 1.2x, each * p < 0.05).

Article Snippet: Primary antibodies (all obtained from Thermo Fisher) were used in blocking reagents (5%-milk-TBS-T for anti-CD44 and anti-CXCR4; 0,5%-milk-TBS-T for actin; 10mL of AdvanBlock-Chemi Blocking Solution (Advansta, Menlo Park, CA, USA) in concentrations as follows: mouse anti-CD44 (Catalog # MA5–13890; 1:133; 1.5 µg/mL) mouse anti-CXCR4 (Catalog # 35-8800; 1:250; 2 µg/mL) and mouse anti- FLI1 (Catalog # MA1-196;1:500; 2 µg/mL) and mouse anti-actin (A1978, Sigma-Aldrich, St. Louis, MO, USA; 1:500) as control.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Role of C-X-C Chemokine Receptor Type 4 (CXCR4) in Cell Adherence and Spheroid Formation of Human Ewing’s Sarcoma Cells under Simulated Microgravity

doi: 10.3390/ijms20236073

Figure Lengend Snippet: This table shows gene expression alterations of genes tested after 24 h of s-µg. The comparative CT (ΔΔCT) method was used for relative quantification of transcription levels, with the control group set as 100%. Each value is displayed as the x-fold of the corresponding expression level of the control group. Upward pointing arrow (↑) symbolizes increase in gene expression and downward pointing arrow (↓) symbolizes decreased gene expression of the respective gene, equal sign (=) means no changed gene expression (values between 0.9x-1.1x). Values marked with an asterisk have shown to be significant ( p ≤ 0.05)

Article Snippet: Primary antibodies (all obtained from Thermo Fisher) were used in blocking reagents (5%-milk-TBS-T for anti-CD44 and anti-CXCR4; 0,5%-milk-TBS-T for actin; 10mL of AdvanBlock-Chemi Blocking Solution (Advansta, Menlo Park, CA, USA) in concentrations as follows: mouse anti-CD44 (Catalog # MA5–13890; 1:133; 1.5 µg/mL) mouse anti-CXCR4 (Catalog # 35-8800; 1:250; 2 µg/mL) and mouse anti- FLI1 (Catalog # MA1-196;1:500; 2 µg/mL) and mouse anti-actin (A1978, Sigma-Aldrich, St. Louis, MO, USA; 1:500) as control.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Role of C-X-C Chemokine Receptor Type 4 (CXCR4) in Cell Adherence and Spheroid Formation of Human Ewing’s Sarcoma Cells under Simulated Microgravity

doi: 10.3390/ijms20236073

Figure Lengend Snippet: ( a ) Protein accumulation of EWS/FLI1 was significantly increased in adherent cells under s-µg compared to the control group (1.6x, * p < 0.05), the bar graph shows the average density of the blots from the respective experimental group (controls, adherent cells under simulated microgravity, spheroids) with the control group defined as 100%. Displayed below there are the corresponding western blot bands (molecular weight 68 kD). The protein of a corresponding independent experiment (1–5) is blotted next to each other as a triplet containing the corresponding 1 g control (C), adherent cells under s-µg (A), and spheroids (S). ( b ) In spheroids and adherent cells s-µg lead to a highly significant decrease in the protein accumulation of the standard CD44s-isoform at approximately 82 kDa (0.2x, 0.6x, ** p < 0.01) which was also significant comparing both groups directly with each other (* p < 0.05). The bar graph shows the average density of the blots from the respective experimental group (controls, adherent cells under simulated microgravity, spheroids): Below the corresponding western blot bands are shown (molecular weight 82 kD). The lighter band at approximately 37kDa corresponds to the unglycosylated CD44 core-protein. The protein of a corresponding independent experiment (1–5) is blotted next to each other as a triplet containing the corresponding 1 g control (C), adherent cells under s-µg (A), and spheroids (S). ( c ) CXCR4 protein accumulation was not significantly altered after 24 h of s-µg, the bar graph shows the average density of the blots from the respective experimental group (controls, adherent cells under simulated microgravity, spheroids) with the control group defined as 100%. Below the corresponding western blot bands were located at a molecular weight of 45 kD. The protein of a corresponding independent experiment (1–5) is blotted next to each other as a triplet containing the corresponding 1 g control (C), adherent cells under s-µg (A) and spheroids (S).

Article Snippet: Primary antibodies (all obtained from Thermo Fisher) were used in blocking reagents (5%-milk-TBS-T for anti-CD44 and anti-CXCR4; 0,5%-milk-TBS-T for actin; 10mL of AdvanBlock-Chemi Blocking Solution (Advansta, Menlo Park, CA, USA) in concentrations as follows: mouse anti-CD44 (Catalog # MA5–13890; 1:133; 1.5 µg/mL) mouse anti-CXCR4 (Catalog # 35-8800; 1:250; 2 µg/mL) and mouse anti- FLI1 (Catalog # MA1-196;1:500; 2 µg/mL) and mouse anti-actin (A1978, Sigma-Aldrich, St. Louis, MO, USA; 1:500) as control.

Techniques: Western Blot, Molecular Weight