Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a H&E staining shows histological evidence of transformation from normal acini to ADM and to PDAC. Magnification, ×10; Scale bar: 2 mm. b Enlarged view of ADM area in a . Magnification, ×100; Scale bar: 200 μm. Black arrows indicate typical ADM circular structures. c Single antibody immunohistochemical staining shows intense REG3A expression the ADM zone. Magnification, ×10; Scale bar: 2 mm. d Enlarged view of ADM area in c . Black arrows indicate typical ADM stained strongly with REG3A. Magnification, ×100; Scale bar: 200 μm.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Staining, Transformation Assay, Immunohistochemical staining, Expressing

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Bright field images showing an increase in ADM events (depicted by black arrows) in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (Magnification, ×200). TGFα-treated mouse primary acinar cells served as a positive control. b Bar graph showing increase in ADM quantity in 3D culture of mouse and human primary acinar cells during the 5-day REG3B or REG3A or TGFα treatment ( n = 3, 15 fields each group, one-way ANOVA and student’s t -test). c Bright field images in the upper row showing ADM events in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (magnification, ×630). Lower four rows are corresponding confocal immunofluorescence images showing a decrease in AMYLASE protein expression (green) and an increase in CK19 (red) protein in REG3B-induced, REG3A-induced, or TGFα-induced ADM (magnification, ×630. Scale bars: 20 μm). d RT-qPCR analysis showed a decrease in acinar-specific mRNA (Ptf1a, Cpa, and Mist1) and an increase in duct-specific mRNA (Ck19 and Nestin) in mouse and human primary acinar cells after 48 h of REG3B or REG3A treatment. ( n = 3 per group, student’s t -test). Values are represented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Cell Culture, Positive Control, Immunofluorescence, Expressing, Quantitative RT-PCR, Standard Deviation, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Western blot showing increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in the pancreatic tissue of REG3B-treated WT mice with caerulein-induced pancreatitis (WT cae+REG3B) and a moderate increase in TG mice with caerulein-induced pancreatitis (TG cae) ( n = 3, one-way ANOVA). b Western blots demonstrating increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in cultured human primary acinar cells and mouse acinar cell line 266-6 after REG3A or REG3B treatment respectively, for 48 h ( n = 3, student’s t -test). c Western blot showing the reduced expression of phosphorylated ERK, MEK, BRAF, and total KRAS in the 266-6 cell line after Reg3b gene knockdown by siRNA ( n = 3, student t -test). d In the upper panel, LY3009120 inhibited ERK, MEK, BRAF phosphorylation in a dose-dependent manner in 266-6 cell line. In the lower panel, LY3009120 (5 μM) blocked REG3B-induced MEK and ERK phosphorylation ( n = 3, one-way ANOVA). e Upper panel, Trametinib efficiently attenuated ERK phosphorylation in a dose-dependent manner in the AR42J cell line ( n = 3, one-way ANOVA). Lower panel, Trametinib (100 nM) blocked REG3B-induced ERK phosphorylation. f , g Bright field images ( g ) with quantification ( f ) show that Trametinib (100 nM) and LY3009120 (5 μM) treatment hindered REG3B-induced ADM in 3D cultures of mouse primary acinar cells (200×, n = 3, one-way ANOVA). h Confocal microscopy shows that Trametinib (100 nM) and LY3009120 (5 μM) treatment reduced CK19 protein expression (red) and increased AMYLASE protein expression (green) in 3D culture of mouse primary acinar cells (Scale bar: 20 μm). Data are represented as means ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Phospho-proteomics, Confocal Microscopy, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Co-localization of EXTL3 (green) with human REG3A (red) or rodent REG3B (red) in ADM zones derived from human primary acinar cells in 3D culture, and AR42J and 266-6 cell lines in 2D culture by immunofluorescence microscopy. (magnification: ×630, Scale bars: 10 μm). b Co-immunoprecipitation of REG3B and EXTL3 in mouse primary acinar cells, rat AR42J and mouse 266-6 acinar cell lines. Lysate-bead/antibody conjugate mixture was eluted with sample buffer without DTT for 10 min at 50 °C (elution 1). Sample buffer with DTT (100 mM) was added to the pelleted beads from elution 1 and boiled for 5 min (elution 2). c Confocal immunofluorescence microscopy showed that EXTL3 monoclonal antibody treatment effectively blocks REG3B-induced ADM as indicated by increased expression of the acinar marker AMYLASE and decreased expression of the ductal marker CK19. (magnification: ×630, Scale bar: 20 μm). d – f Extl3 siRNA or neutralizing antibody treatment reduced the protein expression of KRAS and phosphorylated ERK, MEK, and BRAF in the presence or absence of REG3B for 48 h. d Western blotting analysis of the knockdown of Extl3 by siRNA (20 nM) in 266-6 cell line, e knockdown of Extl3 by siRNA (20 nM) in the context of the 266-6 cell line treated with REG3B for 48 h, f Western blotting analysis of EXTL3 neutralizing antibody treatment (2 μg/ml) in 266-6 and AR42J cell lines and mouse primary acinar cells treated with or without REG3B for 48 h. Human primary acinar cells were treated for 30 min. TGFα treatment serves as a positive control for ADM induction.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Derivative Assay, Immunofluorescence, Microscopy, Immunoprecipitation, Expressing, Marker, Western Blot, Knockdown, Positive Control

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: REG3B/REG3A binds to its receptor, EXTL3 receptor on the acinar cell membrane, and promotes ADM by activating the downstream RAS-RAF-MEK-ERK signaling pathway, in the absence of oncogenic Kras mutation. Targeting REG3B/REG3A, neutralizing its receptor EXTL3, or inhibiting downstream signaling molecules, such as B-RAF (LY3009120) or MEK1/2 (Trametinib), could interrupt the ADM process and potentially prevent early PDAC carcinogenesis.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Membrane, Mutagenesis

Journal: Medical Microbiology and Immunology

Article Title: Inflammation, the kynurenines, and mucosal injury during human experimental enterotoxigenic Escherichia coli infection

doi: 10.1007/s00430-024-00786-z

Figure Lengend Snippet: Overview plasma markers analyzed in the present study

Article Snippet: Plasma concentrations of regenerating islet-derived protein 3 alpha (Reg3A) were estimated using the Human Reg3A DuoSet ELISA kit (Catalog # DY5940-05; R&D Systems, Minneapolis, MN, USA) (Table ).

Techniques: Concentration Assay, Activation Assay

Journal: Journal of Extracellular Vesicles

Article Title: WJMSC‐derived small extracellular vesicle enhance T cell suppression through PD‐L1

doi: 10.1002/jev2.12067

Figure Lengend Snippet: Decreased plasma ST2 and REG3A levels in aGvHD patients with WJMSC infusion. (a,b) ELISA of ST2 (a) and REG3A (b) in the plasma samples from aGvHD patients with the WJMSC infusion. Left, combined analysis; Middle, low‐dose (1.2 × 10 6 /kg WJMSCs); Right, high‐dose (10 × 10 6 /kg WJMSCs). Two cell infusion time points were indicated by red arrows (a,b). Data are mean ± s.e.m. and analysed by unpaired one‐tailed Student's t‐test (a,b). * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: For detection of IFN‐γ, ST2 and REG3 alpha (REG3A), plates were prepared using Duoset human IFN‐γ, ST2 and REG3A ELISA kit according to the manufacturers’ instructions (R&D systems, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Quantification of REG3A mRNA expression in the skin wounds of normal or diabetic patients with acute injury ( n =5). ( b ) Immunohistochemical analysis of REG3A in the skin wounds from three normal or diabetic patients. The samples from normal patients were taken 1 day (two patients) or 2 days after acute injury, whereas the samples from diabetic patients were taken 5 or 9 or 14 days after acute injury. ( c ) The expression of RegIIIγ mRNA in skin wounds of normal and T1D mice 3 days after aseptic injury ( n =6). ( d ) Immunoblot analysis of RegIIIγ in skin extracts taken from 2 mm surrounding the wound edges of normal and T1D mice. ( e ) Immunohistochemical analysis of RegIIIγ in day-3 skin wounds of normal and T1D mice. ( f ) Quantification of TNF-α and IL-6 mRNA expression in day-3 skin wounds from normal and T1D mice treated with or without 100 μg RegIIIγ ( n =8). ( g ) Quantification of TNF-α and IL-6 production by ELISA in day-3 skin wounds of normal and T1D mice ( n =5). ( h ) Wound healing of normal and T1D mice treated with ( n =7) or without 100 μg RegIIIγ ( n =6). ( i ) Photographs of healing wounds from normal and T1D mice treated as in h . All the mice used in the study without specific notes were C57BL/6 strain, and T1D mice were developed by STZ induction. Long scale bars represent 200 μm. Short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset of b and e . The abbreviations used here are normal (Nor), diabetes mellitus (DM), unwounded (unW), wounded (W) and RegIIIγ (R). * P <0.05, ** P <0.01 and *** P <0.001. P values were determined by two-tail t -tests ( a ) or two-way analysis of variance (ANOVA; c , f – h ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Expressing, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) IL-17 production by ELISA in skin extracts taken from 2 mm skin surrounding the wound edges of normal and T1D mice at indicated times ( n =4). ( b ) Immunoblot of REG3A in NHEKs stimulated by different doses of rhIL-33 for 12 h. ( c ) RegIIIγ and IL-33 production by ELISA in skin extracts taken as in a ( n =4). ( d , e ) Immunohistochemical analyses of IL-33 in day-3 skin wounds of normal and T1D mice ( d ) or skin wounds of normal and diabetic patients taken as in ( e ). Long scale bars represent 200 μm while short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset. ( f ) Immunoblot of RegIIIγ in the skin wounds of T1D mice treated with PBS or rmIL-33. The first two samples were day-3 skin wounds of normal and T1D mice. The other three samples were from the skin wounds of T1D mice treated with rmIL-33 a day before wounding (−1) or 1 or 3 days post wounding. ( g ) Wound healing of normal and T1D mice treated with ( n =7) or without rmIL-33 ( n =6). ( h ) IL-33 production in NHEKs induced by different doses of rhIL-17 for 24 h. ( i ) The production of IL-33 and RegIIIγ in day-3 skin wounds of wild-type (WT) and Il17 −/− mice. ( j ) REG3A production in NHEKs induced by 200 ng ml −1 rhIL-17 before and after IL-33 was silenced. ( k ) Immunoblot of RegIIIγ in skin wounds of WT and Il17 −/− mice treated with PBS or 2 μg rmIL-33. ( l ) IL-33 production induced by 200 ng ml −1 rhIL-17 in NHEKs exposed to 20 mM glucose or mannitol for 24 h. ( m ) Immunofluorescent staining of IL-33 in NHEKs treated as in l . Scale bars represent 25 μm. ( n ) IL-33 production in NHEKs treated with 200 ng ml −1 rhIL-17 in the presence or absence of 20 mM glucose before and after AGE was inhibited by 2 mM aminoguanidine. The abbreviations used here are IL-33 shRNA ( IL-33 sh), glucose (Glu), mannitol (Man), aminoguanidine (AG). * P <0.05, ** P <0.01 and *** P <0.001. P values were determined by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemical staining, Staining, shRNA

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Quantification of Tlr3 mRNA expression in day-3 skin wounds of normal and T1D mice ( n =6). ( b ) Quantification of TNF-α and IL-6 mRNA expression in the skin extracts taken from 2 mm surrounding the wound edges of WT and Tlr3 −/− T1D mice at day 3 ( n =6). ( c ) Wound healing of normal WT ( n =12), normal Tlr3 −/− ( n =8), diabetic WT ( n =12) and diabetic Tlr3 −/− ( n =10) mice. ( d ) Quantification of TNF-α ( n =5) and IL-6 ( n =8) protein by ELISA in the skin extracts taken from 2 mm surrounding the wound edges of WT and Tlr3 −/− T1D mice treated with or without RegIIIγ. ( e ) Quantification of TNF-α and IL-6 protein by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A ( n =3). ( f ) Quantification of TNF-α and IL-6 protein by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) in the presence or absence of different mutants of REG3A ( n =3). ( g ) Quantification of TNF-α and IL-6 protein by ELISA in adult human epidermal keratinocytes (AHEKs) treated with 10 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A or C-REG3A ( n =3). * P <0.05, ** P <0.01 and *** P <0.001. NS, no significance; ND, not detected. P values were analysed by one-way analysis of variance (ANOVA; f , g ) or two-way ANOVA ( a – e ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Immunoblot of SHP-1 in NHEKs stimulated with 30 nM REG3A for indicated times. ( b ) Immunoblot of SHP-1 in AHEKs stimulated with different doses of REG3A for 24 h. ( c ) Immunoblot of SHP-1 in mouse skin intradermally injected with 100 μg RegIIIγ for indicated times. ( d , e ) Immunoblot of SHP-1 in NHEKs transfected with different doses of pSG5-C-REG3A ( d ) for indicated times ( e ). ( f ) Immunoblot of SHP-1 in NHEKs stimulated with 100 ng ml −1 rhIL-33 before and after REG3A silencing. ( g ) TNF-α and IL-6 mRNA expression in NHEKs transfected with 1 μg pCMV vector or pCMV-SHP-1 in the presence or absence of 5 μg ml −1 poly(I:C) for 24 h ( n =3). ( h ) TNF-α and IL-6 mRNA expression in NHEKs treated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after SHP-1 silencing ( n =3). ( i ) TNF-α and IL-6 production in AHEKs treated as in h ( n =3). ( j ) TNF-α and IL-6 mRNA expression in day-3 skin wounds of WT normal or T1D mice ( n =5). The mice were intradermally injected with H 2 O or 4 mg of SHP-1 inhibitor SSG before wounding. Three days later, 2 mm skin surrounding the wounds was taken for RNA isolation. The abbreviations used here are SHP-1 shRNA ( SHP-1 sh) and REG3A (R). * P <0.05, ** P <0.01 and *** P <0.001. NS, no significance; ND, not detected. P values were analysed by one-way analysis of variance (ANOVA; g ) or two-way ANOVA ( h – j ). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Western Blot, Injection, Transfection, Expressing, Plasmid Preparation, Isolation, shRNA

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Interaction between Flag-tagged different domains of EXTL3 and REG3A assessed by immunoblot analysis after immunoprecipitation with anti-Flag or anti-REG3A. M: mock; F: full-length EXTL3; N: N-EXTL3 (1–548); C: C-EXTL3 (141–919); ΔNΔC: EXTL3 ΔNΔC (141–548). ( b ) Interaction between REG3A or C-REG3A and Flag-tagged N-EXTL3 assessed by immunoblot analysis after immunoprecipitation with anti-REG3A or anti-Flag. F: full-length REG3A; C: CTLD domain of REG3A. ( c ) Quantification of TNF-α and IL-6 mRNA expression in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after EXTL3 silencing ( n =3). ( d ) Immunoblot of SHP-1 in NHEKs treated with 30 nM REG3A before or after EXTL3 silencing. EXTK3 si: EXTL3 siRNA. ( e ) SHP-1 production in NHEKs treated with 30 nM REG3A in the presence or absence of NF-κB inhibitor (Bay11, 10 μM), Erk inhibitor (PD98059, 20 μM), p38 MAPK inhibitor (SB202190, 5 μM), PI3K inhibitor (LY294002, 50 μM), STAT3 inhibitor (S3I-201,50 μM) and AKT inhibitor (AKT1/2 inhibitor, 8 μM). ( f ) Immunoblot of phosphorylated AKT in NHEKs treated with 30 nM REG3A for 1 h before or after EXTL3 was silenced. ( g ) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or AKT1/2 inhibitor for 1 h. AKT1/2 i: AKT1/2 inhibitor. ( h ) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or STAT3 inhibitor (S3I-201) for 1 h. STAT3 i: STAT3 inhibitor. *** P <0.001. NS, no significance. P values were analysed by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a , b ) Quantification of TNF-α and IL-6 production by ELISA in NHEKs treated with 5 μg ml −1 poly(I:C) ( a ) or in AHEKs treated with 10 μg ml −1 poly(I:C) ( b ) in the presence or absence of JNK inhibitor (SP600125, 15 μM) for 24 h ( n =3). ( c ) The phosphorylation of JNK and c-Jun in NHEKs stimulated by 5 μg ml −1 poly(I:C) in the presence or absence of JNK inhibitor for 1 h. JNK i: JNK inhibitor. ( d , e ) Normalized luciferase activity in 293T cells co-transfected with the plasmid expressing c-Jun (0, 50, 100, 150, 200 ng) and plasmids containing TNF-α promoter (50 ng; d ), or TNF-α promoter with mutation in c-Jun binding site (50 ng; d ), or IL-6 promoter (100 ng; e ), or IL-6 promoter with the mutation in c-Jun binding site (100 ng; e ) for 24 h. ( f ) The phosphorylation of JNK and c-Jun in WT primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( g ) The phosphorylation of JNK2 and c-Jun in Jnk1 −/− primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( h ) The phosphorylation of JNK1 and c-Jun in Jnk2 −/− primary murine keratinocytes stimulated by 10 μg ml −1 poly(I:C) for indicated times. ( i , j ) JNK2 phosphorylation in the skin wounds of WT and Tlr3 −/− normal mice ( i ) or T1D mice ( j ). ( k ) Quantification of TNF-α and IL-6 production by ELISA in day-3 skin wounds of WT and Jnk2 −/− normal and T1D mice ( n =6). ( l ) Wound healing of WT ( n =6), Jnk1 −/− ( n =4) and Jnk2 −/− T1D mice ( n =6). ( m , n ) JNK2 phosphorylation in NHEKs stimulated by 5 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A ( m ) or C-REG3A ( n ). ( o ) JNK2 phosphorylation in AHEKs stimulated by 10 μg ml −1 poly(I:C) in the presence or absence of 30 nM REG3A. * P <0.05, ** or ## P <0.01 and ***or ### P <0.001. P value was analysed by one-way analysis of variance (ANOVA; a , b ) or two-way ANOVA ( d , e , k , l ). Data are the means±s.e.m. and representative of two to three independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes

doi: 10.1038/ncomms13393

Figure Lengend Snippet: ( a ) Phosphorylated JNK2 in NHEKs transfected with pCMV vector, pCMV-muSHP-1(C453S) and pCMV-SHP-1 in the presence or absence of 5 μg ml −1 poly(I:C). ( b ) Phosphorylated JNK2 in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A before or after SHP-1 silencing. ( c ) Interaction between SHP-1 and JNK2 in NHEKs stimulated with 5 μg ml −1 poly(I:C) and/or 30 nM REG3A assessed by immunoblot analysis after immunoprecipitation with anti-SHP-1 or anti-JNK2. ( d ) Immunoblot of p-JNK2, SHP-1 and RegIIIγ in the skin extracts taken from 2 mm surrounding the wound edges of WT normal and T1D mice at indicated times. ( e ) Immunoblot of p-JNK2, SHP-1 and RegIIIγ in the skin wounds of WT normal mice injected with IgG or RegIIIγ-neutralizing antibody. ( f ) Immunoblot of p-JNK2 and SHP-1 in the skin wounds of WT normal and T1D mice injected with 100 μg RegIIIγ. ( g ) Wound healing in T1D mice treated with or without 100 μg RegIIIγ before or after SHP-1 was inhibited by its inhibitor SSG ( n =9). ( h ) The schematic graph reflects the interaction between REG3A and TLR3 signalling. After skin injury, IL-33 induces REG3A expression in epidermal keratinocytes. REG3A, in turn, acts on keratinocytes to induce the negative regulator SHP-1 to selectively inhibit TLR3-activated JNK2, thus controlling TLR3-induced inflammation in skin wounds. However, in diabetes, hyperglycaemia inhibits IL-33 expression induced by IL-17. The reduction of IL-33 leads to the decrease in REG3A and SHP-1 but increased TLR3-activated JNK2 phosphorylation, thus exacerbating inflammation in diabetic skin wounds. The abbreviations used here are control (Ctrl), poly(I:C) (P), REG3A (R) and Reg Ab (RegIIIγ antibody). * P <0.05, and *** P <0.001. NS, no significance. P value was analysed by two-way analysis of variance (ANOVA). Data are the means±s.e.m. and representative of two independent experiments.

Article Snippet: The sections were stained with anti-human REG3A antibody (Shanghai Immunogen Biological Technology &Co. LTD, A101122-LZ0098), or anti-mouse RegIIIγ antibody (ABclonal Biotechnology, WG-00077D-K39), or anti-human IL-33 antibody (R&D, AF4810) or anti-mouse IL-33 antibody (R&D, AF3626), and then reprobed with the horseradish peroxidase-conjugated secondary antibody (Neobioscience, China) according to the manufacturer's introduction.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Injection, Expressing, Phospho-proteomics, Control