anti id3  (Sangon Biotech)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway

doi: 10.1167/iovs.66.9.2

Figure Lengend Snippet: ID3 is preferentially expressed in stem/eTA cell populations in the cornea. ( a ) The scRNA-seq identified three epithelial cell types in mouse cornea. ( b ) ID3 expression in limbal/corneal epithelial cells was plotted. The enclosed group represents stem/early TA cell populations. ID3 expression was higher in stem/early TA cell populations than other limbal/corneal epithelial cells. ( c ) Immunofluorescence staining of human cornea and limbus for K15 and ID3 showed that ID3 was predominantly expressed in the limbal basal layer. The arrows point to colocalization of K15 and ID3.

Article Snippet: The following primary antibodies were used: GAPDH (MAB374, 1:1000 dilution; Sigma-Aldrich), ID3 (9837, 1:500 dilution; Cell Signaling Technology), ERK1 (sc-94; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (sc-7383, 1:1000 dilution; Santa Cruz Biotechnology), Akt (4691, 1:1000 dilution; Cell Signaling Technology), p-Akt S473 (4060, 1:1000 dilution; Cell Signaling Technology), EphA2 (AF3035, 1:250 dilution; R&D Systems), and p-EphA2 S897 (6347, 1:1000 dilution; Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway

doi: 10.1167/iovs.66.9.2

Figure Lengend Snippet: Bulk RNA sequencing in human limbal epithelial cells suggests a role for ID3 in regulation of cell migration. ( a ) RT-qPCR demonstrated significant knockdown of ID3 expression with siRNA transfection ( n = 3). ** P < 0.01. ( b ) The PCA plot suggests that variance due to siRNA transfection was much higher than the occurrence of biological variance. ( c ) The heatmap shows dramatic transcriptome changes due to ID3 knockdown. ( d ) DEGs in HLECs transfected with either siID3 or siControl were subjected to GO enrichment analysis, which indicated that cell migration was the most significantly enriched GO term. This suggests a crucial involvement of ID3 in the regulation of limbal epithelial cell migration. The red rectangles indicate terms associated with migration.

Article Snippet: The following primary antibodies were used: GAPDH (MAB374, 1:1000 dilution; Sigma-Aldrich), ID3 (9837, 1:500 dilution; Cell Signaling Technology), ERK1 (sc-94; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (sc-7383, 1:1000 dilution; Santa Cruz Biotechnology), Akt (4691, 1:1000 dilution; Cell Signaling Technology), p-Akt S473 (4060, 1:1000 dilution; Cell Signaling Technology), EphA2 (AF3035, 1:250 dilution; R&D Systems), and p-EphA2 S897 (6347, 1:1000 dilution; Cell Signaling Technology).

Techniques: RNA Sequencing, Migration, Quantitative RT-PCR, Knockdown, Expressing, Transfection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway

doi: 10.1167/iovs.66.9.2

Figure Lengend Snippet: ID3 depletion in a limbal epithelial cell line enhances cell migration. ( a ) Western blot confirming ID3 knockdown and quantification ( n = 3). ( b ) Time-lapse images of representative samples at 0 hour, 3 hours, 6 hours, 9 hours, and 24 hours after scratch wounding. Dashed lines represent approximate borders of the wound edge. ( c ) Quantification of time-dependent wound closure displaying 3-hour intervals of wound closure during a 24-hour scratch wound assay showed that knockdown of ID3 significantly accelerated wound closure ( n = 3). * P < 0.05. Scale bar : 0.5 mm.

Article Snippet: The following primary antibodies were used: GAPDH (MAB374, 1:1000 dilution; Sigma-Aldrich), ID3 (9837, 1:500 dilution; Cell Signaling Technology), ERK1 (sc-94; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (sc-7383, 1:1000 dilution; Santa Cruz Biotechnology), Akt (4691, 1:1000 dilution; Cell Signaling Technology), p-Akt S473 (4060, 1:1000 dilution; Cell Signaling Technology), EphA2 (AF3035, 1:250 dilution; R&D Systems), and p-EphA2 S897 (6347, 1:1000 dilution; Cell Signaling Technology).

Techniques: Migration, Western Blot, Knockdown, Scratch Wound Assay Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway

doi: 10.1167/iovs.66.9.2

Figure Lengend Snippet: ID3 knockdown reveals increased expression of several genes related to migration. ( a ) RT-qPCR examined the relative gene expression changes of select DEGs associated with cell migration. The expression levels in cells transfected with siID3 were normalized to cells transfected with siControl. ( b , c ) Quantification ( b ) and representative images ( c ) at 6-hour migration after scratch wound of cells treated with GI254023X (GIX) showed with MMP9 inhibition and knockdown of ID3 still significantly enhanced cell migration compared to cells treated with siControl+GI254023X, suggesting that ID3 and MMP9 are two parallel pathways in terms of regulation of cell migration ( n = 3). * P < 0.05. Scale bar : 0.5 mm.

Article Snippet: The following primary antibodies were used: GAPDH (MAB374, 1:1000 dilution; Sigma-Aldrich), ID3 (9837, 1:500 dilution; Cell Signaling Technology), ERK1 (sc-94; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (sc-7383, 1:1000 dilution; Santa Cruz Biotechnology), Akt (4691, 1:1000 dilution; Cell Signaling Technology), p-Akt S473 (4060, 1:1000 dilution; Cell Signaling Technology), EphA2 (AF3035, 1:250 dilution; R&D Systems), and p-EphA2 S897 (6347, 1:1000 dilution; Cell Signaling Technology).

Techniques: Knockdown, Expressing, Migration, Quantitative RT-PCR, Gene Expression, Transfection, Inhibition

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway

doi: 10.1167/iovs.66.9.2

Figure Lengend Snippet: ID3 knockdown increases phosphorylation of Akt and ligand-independent activation of EphA2. ( a , b ) After transfection with siControl or siID3, cells were treated with LY294002, an Akt inhibitor ( a ), or EphrinA1 peptide, a ligand of EphA2 that can block the ligand-independent activation of EphA2 ( b ). Protein lysates were subjected to western blot, and densitometry was conducted using Image Studio. It showed that knockdown of ID3 had no significant effect on phosphorylation of ERK. A significant increase of p-Akt, EphA2, and p-EphA2 (S897, a ligand-independent phosphorylation site) in cells transfected with siID3 was detected. Such increases were reversed by LY294002 or EphrinA1 peptide, respectively ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The following primary antibodies were used: GAPDH (MAB374, 1:1000 dilution; Sigma-Aldrich), ID3 (9837, 1:500 dilution; Cell Signaling Technology), ERK1 (sc-94; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (sc-7383, 1:1000 dilution; Santa Cruz Biotechnology), Akt (4691, 1:1000 dilution; Cell Signaling Technology), p-Akt S473 (4060, 1:1000 dilution; Cell Signaling Technology), EphA2 (AF3035, 1:250 dilution; R&D Systems), and p-EphA2 S897 (6347, 1:1000 dilution; Cell Signaling Technology).

Techniques: Knockdown, Phospho-proteomics, Activation Assay, Transfection, Blocking Assay, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibitor of DNA-Binding 3 Is a Novel Regulator of Limbal Epithelial Cell Migration Via the EphA2/Akt Signaling Pathway

doi: 10.1167/iovs.66.9.2

Figure Lengend Snippet: ID3 negatively regulates limbal epithelial cell migration through EphA2/Akt signaling. After transfection with siControl or siID3, cells were treated with LY294002, an Akt inhibitor, or EphrinA1 peptide, a ligand of EphA2 that can block the ligand-independent activation of EphA2. These cells were subjected to scratch wound assays ( n = 9). ( a ) Representative images of scratch wound assays at 6 hours after wounding. Prior to wounding, cells were treated with the PI3K/Akt inhibitor LY294002. Wound closures were quantified using ImageJ. Quantification is shown on the right. ( b ) Representative images of scratch wound assays at 6 hours after wounding. Prior to wounding, cells were treated with EphrinA1 peptide. Quantification is shown on the right. Scale bar : 0.5 mm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The following primary antibodies were used: GAPDH (MAB374, 1:1000 dilution; Sigma-Aldrich), ID3 (9837, 1:500 dilution; Cell Signaling Technology), ERK1 (sc-94; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (sc-7383, 1:1000 dilution; Santa Cruz Biotechnology), Akt (4691, 1:1000 dilution; Cell Signaling Technology), p-Akt S473 (4060, 1:1000 dilution; Cell Signaling Technology), EphA2 (AF3035, 1:250 dilution; R&D Systems), and p-EphA2 S897 (6347, 1:1000 dilution; Cell Signaling Technology).

Techniques: Migration, Transfection, Blocking Assay, Activation Assay