anti ddr1 primary antibody  (Cell Signaling Technology Inc)

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 1 | Erastin inhibits discoidin domain receptor 1 (DDR1) expression in ferroptosis-sensitive bladder cancer (BC) cells, while it shows no effect on ferroptosis-resistant BC cells. (A) DDR1 mRNA expression in TCCSUP and T24 cells with different concentrations of erastin treatment, assessed using RT-qPCR. (B) DDR1 protein levels in TCCSUP and T24 cells after erastin stimulation, assessed using western blotting. N = 3. *p < 0.05 versus 0 μM group, #p < 0.05 versus 10 μM group.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 2 | Cell transfection efficiency after transfection with si-DDR1 and oe-DDR1 in TCCSUP cells and T24 cells. (A) RT-qPCR analysis of DDR1 mRNA expression in TCCSUP cells after DDR1 overexpression and T24 cells upon DDR1 knockdown. (B) Western blot (WB) analysis and quantification of DDR1 protein level in TCCSUP and T24 cells. (C) Representative immunofluorescence (IF) staining and quantification of DDR1 in TCCSUP and T24 cells. N = 3. *p < 0.05 versus control group.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Western Blot, Immunofluorescence, Staining, Control

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 3 | DDR1 inhibits ferroptosis of BC cells. (A) CCK-8 assay of TCCSUP cells treated with erastin, apoptosis inhibitor ZVAD-FMK, necro- ptosis inhibitor necrosulfonamide or ferroptosis inhibitor ferrostatin-1 combined with NC and OE-DDR1. (B) CCK-8 assay of T24 cells treated with erastin, apoptosis inhibitor ZVAD-FMK, necroptosis inhibitor necrosulfonamide or ferroptosis inhibitor ferrostatin-1 combined with NC and OE- DDR1. (C) Levels of glutathione (GSH), malondialdehyde (MDA) and Fe2+ in TCCSUP cells. (D) Levels of GSH, MDA and Fe2+ in T24 cells. (E) RT- qPCR analysis of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and acyl-CoA synthetase long chain family member 4 (ACSL4) expression in TCCSUP cells. (F) RT-qPCR analysis of GPX4, SLC7A11 and ACSL4 expression in T24 cells. (G) WB analysis and quantifi- cation of SLC7A11, zinc finger E-box binding homeobox 1 (ZEB1), E-cadherin, ACSL4, GPX4, yes-associated protein (YAP), p-YAP, neurofibromin 2 (NF2), and p-NF2 expression in TCCSUP and T24 cells. N = 3. *p < 0.05 versus control group. #p < 0.05 versus NC + erastin or si-NC + erastin group.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: CCK-8 Assay, Quantitative RT-PCR, Expressing, Binding Assay, Control

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 5 | DDR1 inhibits ferroptosis of BC cells by regulating homeobox A6 (HOXA6). (A) Left: RT-qPCR analysis of HOXA6 mRNA expres- sion in TCCSUP cells with or without HOXA6 silencing. Right: WB analysis and quantification of HOXA6 protein level in TCCSUP cells. (B) CCK-8 assay of TCCSUP cells. (C) Levels of GSH, MDA and Fe2+ in TCCSUP cells. (D) The expression of DDR1 in TCCSUP cells with or without HOXA6 silencing using RT-qPCR and western blot. N = 3. *p < 0.05 versus OE-DDR1 + erastin group. (E) RT-qPCR and western blot used to measure the expression of HOXA6 in TCCSUP cells with erastion and DDR1 overexpresssion. N = 3. *p < 0.05 versus control. #p < 0.05 versus erastin. $p < 0.05 versus OE-DDR1.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: Quantitative RT-PCR, CCK-8 Assay, Expressing, Western Blot, Control

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 6 | DDR1 targeting HOXA6 facilitates BC growth and inhibits BC ferroptosis in vivo. (A) Nude mice were xenografted with TCCSUP cells and treated with or without erastin. The subcutaneous xenografts (upper), tumour volumes (lower left) and tumour weights (lower right) are shown. (B) Nude mice were xenografted with T24 cells and treated with or without erastin. The subcutaneous xenografts (upper), tumour volumes (lower left) and tumour weights (lower right) are shown. (C) Levels of tumoural GSH, MDA and Fe2+ in the TCCSUP xenograft group. (D) Levels of tumoural GSH, MDA and Fe2+ in the T24 xenograft group. N = 3. *p < 0.05 versus control group. #p < 0.05 versus erastin group. $p < 0.05 versus OE- DDR1 + erastin group.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: In Vivo, Control

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 8 | Effect of DDR1 on the levels of proteins involved in ferroptosis, EMT and NF2–YAP pathway in T24 xenografts. Representative IHC staining and quantification of GPX4, SLC7A11, ACSL4, ZEB1, E-cadherin, p-YAP and p-NF2 in T24 xenograft group tumours. N = 3. *p < 0.05 versus control group. *p < 0.05 versus control group. #p < 0.05 versus erastin group. $p < 0.05 versus OE-DDR1 + erastin group.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: Immunohistochemistry, Control

Journal: Journal of cellular and molecular medicine

Article Title: DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.

doi: 10.1111/jcmm.70410

Figure Lengend Snippet: FIGURE 9 | Schematic diagram of our study. The ferroptosis inducer erastin inhibited the viability of ferroptosis-sensitive TCCSUP cells, pro- moted ferroptosis and downregulated DDR1 while upregulating HOXA6. Additionally, DDR1 overexpression regulated ferroptosis-related markers (upregulation of GSH, GPX4 and SLC7A11 expression, and down-regulation of MDA, Fe2+ and ACSL4 levels) and EMC-related markers (downreg- ulation of E-cadherin and upregulation of ZEB1), and inhibited YAP–NF-2 signalling pathway activation, promoting the viability of TCCSUP cells and inhibiting ferroptosis, thereby facilitating bladder cancer progression. HOXA6 may be a downstream target of DDR1, and DDR1 may negatively regulate HOXA6. HOXA6 knockdown reversed the effects of DDR1 overexpression on ferroptosis in TCCSUP cells. Yellow arrow: upregulation or promotion, green arrow: downregulation or inhibition and ?: there may be other underlying mechanisms between the two.

Article Snippet: The membranes were blocked with 5% skim milk at 37°C for 1 h and then incubated using primary antibodies against DDR1 (#SC- 374618, 1:1000, Santa Cruz Biotechnology, Dallas, USA), solute carrier family 7 member 11 (SLC7A11, #Ab175186, 1:1000, Abcam, Cambridge, UK), yes- associated protein (YAP, #13584- 1- AP, 1:2000, Proteintech, Rosemont, USA), p- YAP (#29018- 1- AP, 1:2000, Proteintech), acyl- CoA synthetase long chain family member 4 (ACSL4, #Ab155282, 1:10000, Abcam), zinc finger E- box binding homeobox 1 (ZEB1, #Sc- 515797, 1:1000, Santa Cruz Biotechnology), Ecadherin (#20874- 1- AP, 1:20000, Proteintech), glutathione peroxidase 4 (GPX4, #Ab125066, 1:1000, Abcam), neurofibromin 2 (NF2, #21686- 1- AP, 1:2000, Proteintech), p- NF2 (#28851- 1- AP, 1:1000, Proteintech), HOXA6 (#18210- 1- AP, 1:1000, Proteintech) and β- actin (#66009–1- Ig, 1:20000, Proteintech) overnight at 4°C.

Techniques: Over Expression, Expressing, Activation Assay, Knockdown, Inhibition