Journal:

Article Title: Neutrophil gelatinase-associated lipocalin mediates 13- cis retinoic acid-induced apoptosis of human sebaceous gland cells

doi: 10.1172/JCI33869

Figure Lengend Snippet: Immunohistochemistry using an antibody to NGAL was performed on skin sections taken at baseline and at 1 week of isotretinoin treatment. Sections were incubated overnight with a 1:50 dilution of mouse monoclonal LCN2/NGAL antibody and developed using AEC chromagen (red). All sections were counterstained with hematoxylin. Representative images at baseline and after 1 week isotretinoin from Patients 1 and 2 are shown. An image of the epidermis after isotretinoin treatment from Patient 2 is shown. NGAL was expressed in the sebaceous gland and duct of samples of skin taken at 1 week of isotretinoin therapy. NGAL was not expressed in the epidermis. The amount of NGAL staining varies among patients and individual patient results are shown in Table ​Table4.4. Negative control consists of normal human skin incubated with normal mouse IgG1 antibody. Original magnification, ×100.

Article Snippet: Sections were incubated overnight with mouse monoclonal LCN2 antibody (Abcam Inc.).

Techniques: Immunohistochemistry, Incubation, Staining, Negative Control

Journal:

Article Title: Neutrophil gelatinase-associated lipocalin mediates 13- cis retinoic acid-induced apoptosis of human sebaceous gland cells

doi: 10.1172/JCI33869

Figure Lengend Snippet: SEB-1 sebocytes were treated with vehicle as a control (C), 13-cis RA (0.1 nM, 1 nM, 10 nM, 0.1 μM, or 1 μM) or ATRA (0.1 nM, 1 nM, 10 nM, 0.1 μM, or 1 μM) for 48 and 72 hours. (A) LCN2 mRNA expression was verified by QPCR after 48 hours of retinoid treatment. Data represent mean ± SEM of the fold change in gene expression as determined by QPCR of 5 independent samples. Statistical analysis was performed with REST-XL software program and considered significant if *P < 0.05. (B) Protein expression was verified by western blot at 72 hours of retinoid treatment. Blots were incubated with primary antibody to NGAL as well as β-actin as a loading control. Blots were analyzed by densitometry and normalized to β-actin. The graph represents normalized fold-change values (mean ± SEM) relative to control for a minimum of 3 independent blots. Statistical analysis was performed with paired t test; *P < 0.05.

Article Snippet: Sections were incubated overnight with mouse monoclonal LCN2 antibody (Abcam Inc.).

Techniques: Expressing, Software, IF-P, Western Blot, Incubation

Journal:

Article Title: Neutrophil gelatinase-associated lipocalin mediates 13- cis retinoic acid-induced apoptosis of human sebaceous gland cells

doi: 10.1172/JCI33869

Figure Lengend Snippet: SEB-1 sebocytes (2 × 106 cells per 100 μl reaction mixture) were nucleofected with 1 μg siCONTROL, GAPDH (as a specificity control), or LCN2 siRNA. 13-cis RA (0.1 μM) was added 24 hours after nucleofection, and cells were incubated for 48 and 72 hours. Extent of siRNA knockdown of gene expression was verified by QPCR and western blotting for LCN2 and GAPDH after 13-cis RA treatment. GAPDH and LCN2 gene expression were successfully inhibited in their respective samples. (A) QPCR analysis of LCN2 and GAPDH mRNA levels at 48 hours of 0.1 μM 13-cis RA treatment. The expression of LCN2 mRNA was successfully decreased 15-fold by the LCN2 siRNA compared with siCONTROL, whereas expression of GAPDH was minimally affected by siRNA to LCN2. GAPDH mRNA expression was decreased 4-fold by the specific GAPDH siRNA when compared with siCONTROL, whereas siRNA to GAPDH had minimal effects on expression of mRNA for LCN2. Data represent mean ± SEM; n = 6. *P < 0.05 as determined by REST-XL program. (B) Western analysis of GAPDH and NGAL protein levels. NGAL protein expression is decreased to undetectable levels by western blotting at 48 and 72 hours of 0.1 μM 13-cis RA treatment with LCN2 siRNA. Representative blots of 4 independent experiments are shown.

Article Snippet: Sections were incubated overnight with mouse monoclonal LCN2 antibody (Abcam Inc.).

Techniques: Incubation, Expressing, Western Blot

Journal:

Article Title: Neutrophil gelatinase-associated lipocalin mediates 13- cis retinoic acid-induced apoptosis of human sebaceous gland cells

doi: 10.1172/JCI33869

Figure Lengend Snippet: Under siRNA conditions described in Figure ​Figure6,6, immunoblotting revealed that siRNA to LCN2 decreases expression of cleaved caspase 3 by approximately 5-fold compared with siRNA control. Blots were incubated with primary antibodies to cleaved caspase 3 as well as β-actin as a loading control to normalize densitometry values. Three independent scrambled siCONTROL (SCRAM) and LCN2 western blots are shown. p17 and p19 are the cleaved or active fragments of caspase 3. Graph represents the normalized expression of cleaved caspase 3 from 5 independent western blots. These data indicate that NGAL mediates the apoptotic response of SEB-1 sebocytes to 13-cis RA. Statistical analysis was performed with paired t test, mean ± SEM; *P < 0.05.

Article Snippet: Sections were incubated overnight with mouse monoclonal LCN2 antibody (Abcam Inc.).

Techniques: Western Blot, Expressing, Incubation

Journal: Redox Report : Communications in Free Radical Research

Article Title: Melittin alleviates sepsis-induced acute kidney injury by promoting GPX4 expression to inhibit ferroptosis

doi: 10.1080/13510002.2023.2290864

Figure Lengend Snippet: Effect of Melittin Attenuates sepsis-AKI. (a) H&E staining of kidney tissues from the Different groups. The red arrows denote the occurrence of tubular dilatation. The blue arrows highlight the swelling of renal tubular epithelial cells. Scale bars: 400 μm. (b) Tubular injury score. (c) Immunohistochemical staining of kidney with primary antibodies against NGAL. The areas marked with red arrows are NGAL-positive regions. Scale bars: 400 μm. (d) Average percentage of positive staining for NGAL per field.(n = 3). (e) Western blotting of NGAL in kidney tissues. (f) Plasma BUN levels. (g) Plasma creatinine levels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, no significant difference between control and LPS group.

Article Snippet: Incubation was performed with the primary antibodies anti-NGAL antibody (PB9609, Boster, China) and anti-4-HNE antibody (AO2685-4, Boster, China).

Techniques: Staining, Immunohistochemical staining, Western Blot, Clinical Proteomics, Control

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: The Journal of Biological Chemistry

Article Title: Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17 *

doi: 10.1074/jbc.M115.685644

Figure Lengend Snippet: Alignment of immunizing peptides for SLC22A17 antibodies used with human, mouse, and rat SLC22A17 Amino acids that vary between species are in bold.

Article Snippet: The rabbit polyclonal antibodies α-NT-24p3-R and α-CT-24p3-R against rat LCN2-R (rLCN2-R were custom made by ImmunoGlobe GmbH (Himmelstadt, Germany) and have been described elsewhere (see ) ( ).

Techniques:

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: ( A ) Real time PCR analysis of expression of LCN2 in SCC13 and SRB12 cells that were transduced to express Tcf7l1 and its various mutants. ( B ) Real time PCR analysis of Lcn2 in enriched epithelial cells from mouse normal skin, DMBA/TPA-induced papilloma and SCC. ( C ) Real time PCR analysis of LCN2 in SRB12 cells that were transduced to express shRNA against nonspecific sequence (shNS) or two different sets of shRNAs against LCN2 . ( D ) Western analysis of LCN2 in SRB12 cells that were transduced to express empty vector control or Tcf7l1 with shRNA against nonspecific sequence or two different sets of shRNAs against LCN2 . Drug selected transduced cells expressing shRNAs were grafted onto NSG mice and tumors were isolated at the end of eight weeks. ( E ) Quantification of tumor mass. Data are presented as box and whisker plots where boxes span first and third quartiles, bars as the median values, and whiskers as minimum and maximum of all data. *p<0.05, **p<0.01, ***p<0.001 (One-way ANOVA with Dunnett’s post-hoc test). n = 5 (shNS+vector), n = 4 (sh LCN2 #1+vector), n = 2 (sh LCN2 #2+vector), n = 3 (shNS+ Tcf7l1 ), n = 3 (sh LCN2 #1+ Tcf7l1 ), n = 3 (sh LCN2 #2+ Tcf7l1 ). ( F ) Representative images of tumors. ( G ) Quantification of cell migration in transwell assay. Human SCC (SRB12) cells with tet-inducible Tcf7l1 were treated with doxycycline for 48 hr and their migratory ability was measured using the Boyden chamber-based cell migration assay. Cells were allowed to migrate toward the lower compartment containing media with 10% FBS as chemoattractant, supplemented with neutralizing antibody against LCN2 or mouse IgG isotype control. After 30 hr, migrated cells were fixed by 4%PFA, counterstained with crystal violet, and quantified. Data are presented as mean ± s.d. **p<0.01 (One-way ANOVA with Dunnett’s post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.23242.018

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, shRNA, Sequencing, Western Blot, Plasmid Preparation, Control, Isolation, Whisker Assay, Migration, Transwell Assay, Cell Migration Assay

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: ( A ) Immunofluorescence images of LCN2 staining in control and TCF7L1-induced skin. Control or tet-inducible Tcf7l1 mice were on a doxycycline containing diet for five days and their skins were immunostained for LCN2 (green) and basement membrane marker β4-integrin (red). ( B ) Images of immunohistochemical staining of LCN2 in normal mouse skin, and DMBA/TPA-induced papilloma and skin SCC. Bar denotes 100 µm. DOI: http://dx.doi.org/10.7554/eLife.23242.019

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Immunofluorescence, Staining, Control, Membrane, Marker, Immunohistochemical staining

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: ( A ) Representative images of negative and positive immunohistochemical staining of TCF7L1 and LCN2 in human skin SCC (middle and bottom panels) and adjacent normal skin (top). Bar denotes 100 µm. ( B ) Graph summarizing the expression of TCF7L1 and LCN2 in human skin SCC samples. ( C ) Graph summarizing the expression of LCN2 in TCF7L1-subgrouped human skin SCC samples (n = 16). DOI: http://dx.doi.org/10.7554/eLife.23242.020

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: eLife

Article Title: TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2

doi: 10.7554/eLife.23242

Figure Lengend Snippet: Xenografted tumors (n = 3) expressing empty vector or vector expressing tet-inducible Tcf7l1 with or without sh LCN2#1 were dissociated and analyzed by flow cytometry for CD11b+Ly6G+Ly6C+ (neutrophils) and CD11b +F4/80+ (macrophages). Representative plots showing the gating of ( A ) CD11b+Ly6G+Ly6C+ and ( B ) CD11b +F4/80+. Graph quantifying the percentage of total cells that express ( C ) CD11+Ly6G+Ly6C+ and ( D ) CD11b +F4/80+. Data are presented as mean ± s.d. **p<0.05 (One-way ANOVA with Dunnett’s post-hoc test). DOI: http://dx.doi.org/10.7554/eLife.23242.021

Article Snippet: Rabbit anti-Myc epitope (1:1,000, Cell Signaling Technology #2278 RRID: AB_490778 ), guinea pig anti-TCF7L1 (1:1,000, lab-generated), rabbit anti-TCF7L2 (1:1,000, Cell Signaling #2569S RRID: AB_2199816 ), rabbit anti-human LCN2 (1:1,000, Abcam #ab125075 RRID: AB_10978084 ), mouse anti-β-actin (1:5,000, Sigma #A5441 RRID: AB_476744 ).

Techniques: Expressing, Plasmid Preparation, Flow Cytometry