Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Control, Molecular Weight, Marker

Journal: Cell Death and Differentiation

Article Title: Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration

doi: 10.1038/s41418-022-00967-4

Figure Lengend Snippet: Key reagents and resources used in this study.

Article Snippet: TBK1/NAK (D1B4) Rabbit mAb , Cell Signaling Technology , cat#3504.

Techniques: Marker, Recombinant, Transfection, Drug discovery, Gene Expression, Microarray, Software

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Fhl1 and Sdpr expression in normal epithelium and tumors from human breast, kidney, and prostate. Immunohistochemical staining was performed on normal and tumor human tissue. Fhl1 and Sdpr staining was evident in normal epithelial cells of the breast, kidney, and prostate. In contrast, both Fhl1 and Sdpr expression were suppressed in infiltrating mammary duct carcinoma, renal cell carcinoma, and prostate adenocarcinoma. (bar = 60 microns).

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Fhl1 and Sdpr expression in human breast, kidney, and prostate tumors

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Expressing

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Selection of genes affected by Fhl1. Microarray analysis was performed to identify probe sets representing genes with mean fold change differences of 2 or more with P < 0.05 by t‐test. (a) Fhl1 transfected Src transformed cells were compared with control transfectants. Of approximately 45 000 probe sets examined, genes represented by 459 were induced by Fhl1, whereas genes represented by 189 were suppressed. (b) Non‐transformed cells were compared with Src transformed cells to identify genes affected by Fhl1 that were correspondingly regulated by Src. Genes represented by 53 probe sets were induced by Fhl1 and suppressed by Src, whereas 48 were suppressed by Fhl1 and induced by Src. (c) Src‐transformed homozygous null Cas knockout cells transfected with Cas were compared with control transfectants to identify genes correspondingly regulated by Fhl1, Scr, and Cas. Genes represented by four probe sets were induced by Fhl1 and suppressed by Src and Cas, whereas genes represented by two probe sets were suppressed by Fhl1 and induced by Src and Cas.

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Selection, Microarray, Transfection, Transformation Assay, Control, Knock-Out

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Effects of Src and mitogen‐activated protein kinase (MAPK) activity on Fhl1 and Sdpr expression. (a) An equal amount of total protein (10 µg/lane) from non‐transformed and Src‐transformed NIH 3T3 cells expressing a Central Dogma (CD)‐tagged Sdpr–GFP fusion protein was analyzed by Western blotting with antiserum specific for green fluorescence protein (GFP), Sdpr, mitogen‐activated protein kinase kinase (MEK), MAPK, active MAPK (p‐MAPK), active Src (p‐Src), and β‐actin as indicated. (b) An equal amount of protein (100 µg) was immunoprecipitated from non‐transformed and Src‐transformed NIH 3T3 cells expressing a CD‐tagged Sdpr–GFP fusion protein, and then examined by Western blot analysis with GFP or Sdpr antiserum. (c) An equal amount of total protein (10 µg/lane) from non‐transformed and Src‐transformed embryonic mouse brain cells was analyzed by Western blotting with antiserum specific for Sdpr, Fhl1, MAPK, active MAPK (p‐MAPK), active Src (p‐Src), and β‐actin as indicated. (d) An equal amount of total protein (10 µg/lane) from non‐transformed and Src transformed embryonic mouse fibroblasts with or without Cas was analyzed by Western blotting with antiserum specific for Fhl1, MAPK, active MAPK (p‐MAPK), Cas, active Src (p‐Src), and β‐actin as indicated. Src‐transformed cells were also treated with the MEK blocker PD98059 to determine if Src requires MAPK to suppress Fhl1 and Sdpr expression. In all cases, more Fhl1 and Sdpr protein was present in non‐transformed cells than Src‐transformed cells independent of MAPK activity.

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Activity Assay, Expressing, Transformation Assay, Western Blot, Fluorescence, Immunoprecipitation

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Correlation of Fhl1 and Sdpr expression with Gleason stage of prostate tumors

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Expressing

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Fhl1 gene methylation in Src transformed cells. The transcriptional start site of Fhl1 (+1) lies in a CpG island of several hundred residues. PCR products from bisulfate‐treated DNA from non‐transformed and Src‐transformed cells were subcloned and sequenced. Data are shown as the percentage of each CpG sequence that was methylated (n = 5). More methylation of most of these sites was found in Src‐transformed cells than in non‐transformed cells.

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Methylation, Transformation Assay, Sequencing

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Genes regulated by Fhl1

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques:

Journal: Cell stem cell

Article Title: A M etformin -R esponsive M etabolic P athway controls distinct steps in gastric progenitor fate decisions and maturation

doi: 10.1016/j.stem.2020.03.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Vectastain Elite ABC HRP Kit , Vector Laboratories , Cat#PK6100.

Techniques: Recombinant, SYBR Green Assay, Protein Extraction, Plasmid Preparation, Microarray, Expressing, Software, Imaging

Journal: Nature immunology

Article Title: In situ neutrophil efferocytosis shapes T cell immunity to influenza infection

doi: 10.1038/s41590-020-0746-x

Figure Lengend Snippet: (a) A cytokine/growth factor antibody microarray was performed with lysates of the influenza-infected (day 4) trachea after neutrophil depletion (1A8). IgG: isotype-treated group. The signals at the three corners are loading controls. (b) EGF secretion by neutrophils in response to TNF (2–200 ng/ml), fMLP (0.1–10 μM), PMA (1–100 nM) or FasL (1 −100 ng/ml) stimulation was determined by Western blot analysis of neutrophil supernatants with an EGF-specific antibody. Each panel shows one representative image of three replicated experiments. (c) FasL-induced EGF secretion in the presence of Brefeldin A or z-VAD-fmk. TCL; total cell lysate. (d) Western blot analysis of EGFR activation (phospho-EGFR; pEGFR) and its downstream signaling protein AKT (phospho-AKT; pAKT) in mouse primary lung epithelial cells treated with increasing concentrations of recombinant EGF (rEGF) or apoptotic neutrophil supernatant. One representative image of three repeated experiments is shown. (e) Flow cytometric analysis of EGFR activation (anti-p-EGFR) in permeabilized monocytes after treatments with increasing concentrations of rEGF or apoptotic neutrophil supernatant. Data are presented as the mean ± SEM, n = 3 mice per group. Statistical differences of rEGF vs. PBS treatment were assessed by nonparametric Mann-Whitney test. (*P < 0.01).

Article Snippet: Antibodies against EGFR and phospho-EGFR for Western blotting were purchased from Cell Signaling Technology (Danvers, MA) and fluorophore-conjugated phospho-EGFR antibody for flow cytometry was purchased from R&D Systems.

Techniques: Microarray, Infection, Western Blot, Activation Assay, Recombinant, MANN-WHITNEY