recombinant mouse hb-egf protein  (ProSpec)

Journal: Cell Research

Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

doi: 10.1038/s41422-020-0309-6

Figure Lengend Snippet: a HUVECs or HaCaT cells were exposed to 15 μM sorafenib for 24 h. The level of s-HBEGF in the supernatant or pro-HBEGF in total cell lysates was detected by western blot. b s-HBEGF concentrations in supernatants of HUVECs were measured by HBEGF peptide enzyme-linked immunosorbent assay (ELISA) ( n = 3). c Representative photos of ten patients with HFSR of various grades. d s-HBEGF concentrations in the serum of ten healthy volunteers and ten patients were measured by ELISA. e HaCaT cells were treated with s-HBEGF for 72 h. Cell survival rate was detected by SRB assay ( n = 3). f RT-qPCR analysis of KRT1 , KRT10 , LORICRIN and IVL in HaCaT cells treated with s-HBEGF recombinant protein for 24 h ( n = 3). g HaCaT cells were treated with CdM CTRL or CdM SORA in the presence of HBEGF neutralization antibody for 24 h. The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were detected by RT-qPCR ( n = 3). h HUVECs were transfected with scramble shRNA or HBEGF shRNA via lentivirus. The level of s-HBEGF in the supernatant or pro-HBEGF in total cell lysates was detected by western blot. i CdM CTRL or CdM SORA was collected from HUVECs transfected with scramble shRNA or HBEGF shRNA. HaCaT cells were treated with CdM CTRL or CdM SORA for 24 h. The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were detected by RT-qPCR ( n = 3). j – l Mice were treated with HBEGF neutralization antibody (100 ng/mouse) twice a week by i.v. and/or sorafenib (100 mg/kg) daily by i.g. for 30 days ( n = 5/group). j Representative H&E staining and KRT5, KRT1, LORICRIN immunohistochemistry staining were performed on the paws of mice. Scale bar, 50 μm. k Quantitative analysis of epidermal hyper-keratosis assessed by measuring the stratum corneum thickness ( n = 5/group). l s-HBEGF concentrations in the serum of each mouse were measured by ELISA ( n = 5/group). Densitometric values are shown as optical density after ACTB normalization using Image J. Horizontal bars in ( d ), ( k ) and ( l ) represent mean values. The results in ( b ), ( e ), ( f ), ( g ) and ( i ) are presented as the mean ± SD. Statistical analyses were performed using unpaired two-tailed Student’s t test in ( b ), ( d ), ( g ) and ( i ). Statistical analyses were performed using one-way ANOVA with Dunn’s post hoc test when comparing the levels of KRT1 and IVL and with LSD post hoc test when comparing the levels of KRT10 and LORICRIN in ( f ) and with LSD post hoc test in ( k ) and ( i ). * P < 0.05; ** P < 0.01; *** P < 0.001. SORA sorafenib, CdM HUVECs conditional medium, CTRL control.

Article Snippet: Mouse HBEGF recombinant protein (Novus Biologicals, NBP2-35069-10 ug) was used in vivo (25 ng per mouse).

Techniques: Western Blot, Peptide ELISA, Enzyme-linked Immunosorbent Assay, Sulforhodamine B Assay, Quantitative RT-PCR, Recombinant, Neutralization, Transfection, shRNA, Staining, Immunohistochemistry, Two Tailed Test, Control

Journal: Cell Research

Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

doi: 10.1038/s41422-020-0309-6

Figure Lengend Snippet: a HaCaT cells were transfected with non-targeting siRNA or siRNA targeting EGFR . The transcription level of EGFR was detected by RT-qPCR (upper panel, n = 3) and the expression level of EGFR was determined by western blot (lower panel). b – e HaCaT cells were transfected with non-targeting siRNA or siRNA targeting EGFR , followed by treatment with CdM CTRL or CdM SORA for 24 h ( b , d ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( c , e ). The cell survival rates were measured by SRB assay ( n = 3) ( b , c ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( d , e ). f Relevant EGFR downstream signaling pathways were examined by western blot. g Human primary keratinocytes were treated with s-HBEGF and the expression levels of p-JNK1/2, JNK2, p-JNK1 and JNK1 were assessed by western blot. h HaCaT cells or human primary keratinocytes were treated with or without sorafenib, CdM CTRL or CdM SORA . The expression levels of p-JNK1/2, JNK2, p-JNK1 and JNK1 were assessed by western blot. i HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 . JNK2 transcription level was detected by RT-qPCR (upper panel, n = 3) and the expression level of JNK2 was determined by western blot (lower panel). j–m HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 , followed by treatment with CdM CTRL or CdM SORA for 24 h ( j , l ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( k , m ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( j , k ). The cell survival rates were measured by SRB assay ( n = 3) ( l , m ). Densitometric values are shown as optical density after ACTB or GAPDH normalization using Image J. The results in ( a ), ( b ), ( c ), ( d ), ( e ), ( i ), ( j ), ( k ), ( l ) and ( m ) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with Dunn’s post hoc test in ( a ), ( i ) and when comparing the levels of KRT1 in ( j ) and with LSD post hoc test in ( d ), ( e ), ( k ) and when comparing the levels of KRT10 , LORICRIN and IVL in ( j ). n.s. no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. SORA sorafenib, CdM HUVECs conditional medium, CTRL control.

Article Snippet: Mouse HBEGF recombinant protein (Novus Biologicals, NBP2-35069-10 ug) was used in vivo (25 ng per mouse).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Sulforhodamine B Assay, Protein-Protein interactions, Control

Journal: Cell Research

Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

doi: 10.1038/s41422-020-0309-6

Figure Lengend Snippet: a HaCaT cells or human primary keratinocytes were treated with or without sorafenib, CdM CTRL or CdM SORA . The expression level of SIRT1 was detected by western blot. b HaCaT cells or human primary keratinocytes were treated with s-HBEGF for 24 h. The expression level of SIRT1 was analyzed by western blot. c Representative western blot indicated the expression of SIRT1 in the stratum corneum of mice in control or sorafenib-treated group ( n = 5/group). d Representative western blot indicated the expression of SIRT1 in the stratum corneum of mice in control or s-HBEGF-treated group ( n = 5/group). e Representative immunohistochemistry images showing SIRT1-stained paws of mice in control, sorafenib, HBEGF neutralizing antibody or combination group (left panel). Representative immunohistochemistry images showing SIRT1-stained paws of mice with or without s-HBEGF treatment (right panel). Scale bar, 50 µm. f HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 , followed by treatment with or without s-HBEGF (2.5 ng/mL) for 24 h. The expression levels of p-JNK1/2, JNK2 and SIRT1 were determined by western blot. g HaCaT cells were treated with s-HBEGF for 24 h. The transcription level of SIRT1 was measured by RT-qPCR ( n = 3). h HaCaT cells were treated with CHX with or without s-HBEGF at different time points, and SIRT1 protein level was measured by western blot. i HaCaT cells were treated with s-HBEGF for 24 h. The expression levels of p-SIRT1 and SIRT1 were analyzed by western blot. j Human primary keratinocytes were treated with s-HBEGF for 24 h. The expression levels of p-SIRT1 and SIRT1 were analyzed by western blot. k HaCaT cells were treated with s-HBEGF for 24 h. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and probed with anti-Ub antibody or with anti-SIRT1 antibody. Protein expression levels of endogenous p-SIRT1 and SIRT1 are displayed. l HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 , followed by treatment with or without s-HBEGF (2.5 ng/mL) for 24 h. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and probed with anti-Ub antibody or with anti-SIRT1 antibody. Protein expression levels of endogenous p-SIRT1, SIRT1, p-JNK1/2, JNK2 are displayed. Densitometric values are shown as optical density after ACTB or GAPDH normalization using Image J. The results in ( g ) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with LSD post hoc test in ( g ). n.s. no significance. SORA sorafenib, CHX cycloheximide, CdM HUVECs conditional medium, CTRL control, IP immunoprecipitant, WCL whole cell lysate.

Article Snippet: Mouse HBEGF recombinant protein (Novus Biologicals, NBP2-35069-10 ug) was used in vivo (25 ng per mouse).

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Staining, Transfection, Quantitative RT-PCR, Immunoprecipitation

Journal: Cell Research

Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

doi: 10.1038/s41422-020-0309-6

Figure Lengend Snippet: a HaCaT cells were transfected with non-targeting siRNA or siRNA targeting SIRT1 . SIRT1 transcription level was detected by RT-qPCR (upper panel, n = 3) and the expression level of SIRT1 was determined by western blot (lower panel). b – e HaCaT cells were transfected with non-targeting siRNA or siRNA targeting SIRT1 , followed by treatment with CdM CTRL or CdM SORA for 24 h ( b , d ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( c , e ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( b , c ). The cell survival rates were measured by SRB assay ( n = 3) ( d , e ). f – i Mice were randomly divided into 4 groups. After injection of AAV1-sh Sirt1 adeno virus into the paws for 2 weeks, mice were treated with CMC-Na or sorafenib (100 mg/kg) daily by i.g. for 30 days ( n = 5/group). f Representative western blot indicated the expression of SIRT1 in stratum corneum of mice in each group ( n = 3/group). g Representative H&E staining and KRT5, KRT1, LORICRIN, SIRT1 immunohistochemistry staining were performed on the paws of mice. Scale bar, 50 μm. h Quantitative analysis of epidermal hyper-keratosis assessed by measuring the stratum corneum thickness. i s-HBEGF concentrations in the serum of each mouse were measured by ELISA ( n = 5/group). Densitometric values are shown as optical density after GAPDH or ACTB normalization using Image J. Horizontal bars in ( h ) and ( i ) represent mean values. The results in ( a ), ( b ), ( c ), ( d ) and ( e ) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with LSD post hoc test in ( b ), ( c ), ( h ) and ( i ) and with Dunn’s post hoc test in ( a ). n.s. no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. SORA sorafenib, CdM HUVECs conditional medium, CTRL control.

Article Snippet: Mouse HBEGF recombinant protein (Novus Biologicals, NBP2-35069-10 ug) was used in vivo (25 ng per mouse).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Sulforhodamine B Assay, Injection, Virus, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Research

Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

doi: 10.1038/s41422-020-0309-6

Figure Lengend Snippet: a HaCaT cells were treated with or without 10 mM nicotinamide, followed by treatment with CdM CTRL or CdM SORA for 24 h. The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3). b – d Mice were treated with vehicle, sorafenib (100 mg/kg/day), nicotinamide (100 mg/kg/day) or sorafenib plus nicotinamide by intragastric administration for 30 days ( n = 5/group). b Representative histopathology images show H&E-stained paws of the mice in control, sorafenib, nicotinamide or combination group. Representative immunohistochemistry images show KRT5, KRT1 or LORICRIN-stained paws of each group. Scale bar, 50 µm. c Thickness quantification of corneous layer of paws of the mice in control, sorafenib, nicotinamide or combination group ( n = 5/group). d s-HBEGF concentrations in the serum of each mouse were measured by ELISA ( n = 5/group). e Hands and feet of patients with sorafenib-induced HFSR showing hyper-keratosis before (left panel) and after (right panel) administration with 50 or 100 mg nicotinic acid (depending on HSFR grade) three times a day. f Diagnosis information of each patient. Horizontal bars in ( c ) and ( d ) represent mean values. The results in ( a ) are presented as the mean ± SD. Statistical analyses were performed using unpaired two-tailed Student’s t test in ( a ), and one-way ANOVA with LSD post hoc test in ( c ) and ( d ). n.s. no significance; * P < 0.05; *** P < 0.001. NAM nicotinamide, SORA sorafenib, CdM HUVECs conditional medium, CTRL control.

Article Snippet: Mouse HBEGF recombinant protein (Novus Biologicals, NBP2-35069-10 ug) was used in vivo (25 ng per mouse).

Techniques: Quantitative RT-PCR, Histopathology, Staining, Control, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Two Tailed Test

Journal: Cell Research

Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

doi: 10.1038/s41422-020-0309-6

Figure Lengend Snippet: Vascular endothelial cells release s-HBEGF upon sorafenib stimulation. s-HBEGF binds to EGFR and leads to JNK2 phosphorylation in keratinocytes. The activated JNK2 subsequently stabilizes SIRT1, which eventually results in keratinization. The classic SIRT1 inhibitor nicotinamide could effectively reverse sorafenib-induced HFSR.

Article Snippet: Mouse HBEGF recombinant protein (Novus Biologicals, NBP2-35069-10 ug) was used in vivo (25 ng per mouse).

Techniques: Phospho-proteomics