Journal: bioRxiv

Article Title: Astrocytic Regulation of Basal Ganglia Dopamine/D2-Dependent Behaviors

doi: 10.1101/2021.05.11.443394

Figure Lengend Snippet: a. Heat map of 65 inflammatory markers selected by a microarray screening from Dys1+/+ (n24), and Dys1+/- (n25) littermates. The heat map is based on hierarchical clustering of genes involved in inflammation states. All gene expression levels were transformed to scores ranging from −0.5 to 0.5 and were colored blue, white, or red to represent low, moderate, or high expression levels, respectively. The relative expression levels were scaled based on their mean and do not represent expression levels in comparison with controls. Dys1+/- mice show higher expression for these genes compared to Dys1+/+ littermates (t-test: t 128 =-2.23, p=0.028). *p<0.05 vs Dys1+/+. b. Quantification of cumulative GFAP intensity from confocal images from PFC, NAcc, STR and GPe displayed by Dys1+/+ and Dys1+/- littermates (n4/genotype, 1/brain region averaged from 9 samples each). Scale bars, 20μm (t-test: t 31 =-2.32, p=0.027) *p<0.05 vs Dys1+/+. c. Representative confocal images of GFAP positive astrocytes in the analyzed brain regions. d. Transmission electron microscopy (TEM) images of the Golgi Complex (GC) in neurons and astrocytes in Dys1+/+ and Dys1+/- littermates. Surface density of GC (SvGC/SvCyt) in Dys1+/- mice was significantly higher than in Dys1+/+ littermates (neurons t 21 =-2.70, p=0.013; astrocytes t 12 =-4.40, p=0.0009). *p<0.05, **p<0.001 vs Dys1+/+. e. Maximum intensity projections of ventral ganglion cells, from Drosophila third instar larvae expressing UAS-GalT-GFP to visualize Golgi cisternae, for controls (tubulin-Gal4/+) and UAS-Dysb RNAi. Tissues were labeled with anti αRepo antibody to visualize glial cells. Scale bar 20 µm. On the right TEM images of third instar larvae brain showing the Golgi apparatus in the neuronal cell bodies of ventral ganglion for the above genotypes. Flies expressing UAS-RNAi Dybs ubiquitously showed swelling of largely inflated Golgi cisternae (arrows). Scale bar 500 nm. f. Quantification of the glial nuclei distribution in control (tubulin - Gal4 /+ ) and RNAi Dysb/tubulin - Gal4 flies. g. Representative western blots and densitometric analysis of Dys1A (50 kDa) and Dys1C (38 kDa) isoforms. β-actin used as loading control. In brain lysate of adult P90 mice both isoforms were revealed, with higher expression for Dys1C compared to Dys1A (t-test: t 10 =-5.77, p=0.0002). Dys1A was the only isoform expressed in glial cells (t-test: t 20 =-6.32, p < 0.0001). Similar to brain lysate, neuronal cultures show the expression of both isoforms with relative higher levels of Dys1C (t-test: t 10 =-2.57, p=0.02). h. Astrocytes cultures at different developmental time points (day 7=DIV7; day 14= DIV14; day 21= DIV21) confirming no expression of Dys1C in astrocytes. i. Neuronal cultures at different developmental time points (DIV7, 14 and 21) showing relative higher expression of Dys1C compared to Dys1A. Bar graphs show mean±s.e.m.

Article Snippet: Subsequently they were incubated overnight at 4 °C with 1:300 rabbit polyclonal anti-GFAP antibody (Novus Biologicals, Centennial, CO, USA) in blocking solution.

Techniques: Microarray, Gene Expression, Transformation Assay, Expressing, Comparison, Transmission Assay, Electron Microscopy, Labeling, Control, Western Blot

Journal: bioRxiv

Article Title: Astrocytic Regulation of Basal Ganglia Dopamine/D2-Dependent Behaviors

doi: 10.1101/2021.05.11.443394

Figure Lengend Snippet: a. Experimental design and timeline to generate reduction (+/-) or absence (-/-) of Dys1A in astrocytes of adult mice subsequently subjected to molecular and behavioral evaluation. Dys1Afloxed mice were bred with conditional Glast CreErT2 Tomato + mice, and offspring were treated with tamoxifen at post-natal days 60-61 to then be tested between post-natal days 90-120. b. Experimental design, and gating strategy to FACS-sorted astrocytes for subsequent RT-qPCR analyses. c. Relative mRNA expression of Glast assessed by RT-qPCR in tdTomato-positive (tdT+) and tdTomato-negative (tdT-) cells sorted from the basal ganglia of Dys1AGlast+/+ (n10) and Dys1AGlast-/- (n13) mice. tdT+ cells show increased expression of Glast compared to tdT- cells (t-test: t=3.25, df=21, p< 0.005). **p<0.005 tdT+ vs tdT- cells. d. Relative mRNA expression of NeuN and Glast assessed by RT-qPCR in tdTomato-positive cells sorted from the basal ganglia of Dys1AGlast+/+ (n10) and Dys1AGlast-/- (n10) mice. tdTomato-positive cells show an equal expression of Glast in Dys1AGlast+/+ and Dys1AGlast-/- mice (Two-way ANOVA; genotype effect: F 1,12 =0.084; p=0.78), but no detectable levels of NeuN (Two-way ANOVA; gene expression: F 1,12 =13.16; p=0.003). **p<0.005 Glast vs NeuN. Expression levels were normalized by Gapdh expression. e. Relative mRNA expression of the Dys1A isoform assessed by RT-qPCR in tdTomato-positive cells sorted through FACS from the basal ganglia of Dys1AGlast+/+ (n8) and Dys1AGlast-/- (n8) mice, showing the abolishment of Dys1A expression from Glast-positive astrocytes in the latter group (One-way ANOVA; F 1,14 =18.32; p=0.0008). **p<0.005 vs Dys1AGlast+/+. Expression levels are normalized by Gapdh expression. Data shown as fold-change compared with Dys1AGlast+/+ control mice. f. Left: representative 10x confocal images of GFAP-stained brain section at the level of GPe, white square indicates the area magnified 20x for subsequent analyses (scale bar 500µm); right: representative 20x confocal images of GPe brain sections from Dys1AGlast+/+ and -/- mice showing Glast-/tdTomato-positive astrocytes and GFAP-immunoreactivity (scale bar 20µm). g. There was no difference in Glast/tdTomato-positive astrocytes density in the GPe (1000 cells x mm 2 ) between Dys1AGlast+/+ (10), Dys1AGlast+/- (6), and Dys1AGlast-/- (7) mice (One-way ANOVA: F 2,20 =2.33; p=0.12). h. Representative images and quantification of GFAP and Glast-positive astrocytes morphology in the GPe of Dys1AGlast+/+ (n6) and Dys1AGlast-/- (n6) mice. No genotype-dependent difference was observed in the astrocytic surface area measured by GFAP immunoreactivity (t-test: t 10 =-0.46, p=0.65). i. Spontaneous distance traveled by Dys1AGlast+/+ (n20), Dys1AGlast+/- (n24), and Dys1AGlast-/- (n11) during 30 minutes exposure to an open field arena. No genotype differences were evident (Two-way repeated measure ANOVA, genotype effect: F 2,52 =2.79; p=0.07; time*genotype effect: F 10,260 =0.91; p=0.53). j. Percent pre-pulse inhibition (PPI) of the 120dB acoustic startle response displayed by Dys1AGlast+/+ (n21), Dys1AGlast+/- (n24), and Dys1AGlast-/- (n13) littermates. Dys1AGlast-/- have reduced PPI compared to Dys1AGlast+/+ mice (Two-way repeated measure ANOVA; genotype effect: F 2,54 =8.59 p=0.0006). ***p<0.0005 vs Dys1AGlast+/+. k. Breakpoint during a food-driven operant behavior test with increasing progressive ratio (PR) displayed by Dys1AGlast+/+ (n15), Dys1AGlast+/- (n15), and Dys1AGlast-/- (n9) littermates. Both Dys1AGlast+/- and Dys1AGlast-/- mice showed lower breakpoints than Dys1AGlast+/+ mice (Two-way repeated measure ANOVA; genotype effect: F 2,36 =4.35; p=0.020). *p<0.05 vs Dys1AGlast+/+. Bar and line graphs show mean ± s.e.m.

Article Snippet: Subsequently they were incubated overnight at 4 °C with 1:300 rabbit polyclonal anti-GFAP antibody (Novus Biologicals, Centennial, CO, USA) in blocking solution.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Control, Staining, Inhibition

Journal: bioRxiv

Article Title: Astrocytic Regulation of Basal Ganglia Dopamine/D2-Dependent Behaviors

doi: 10.1101/2021.05.11.443394

Figure Lengend Snippet: a. Experimental design to selectively delete dopamine D2 receptors of D2 flox/flox mice restricted to GPe-astrocyte, bilaterally. Reconstruction of viral spread across the GPe along anteroposterior axis. Low magnification images of a representative viral spread on a coronal slices of a D2 flox/flox mice injected with AAV-GFAP-Cre-GFP (scale bar=500µm). Findings were replicated in two independent experiments with similar results. b. Percentage PPI of the 120dB acoustic startle response displayed by control mice bilaterally injected in the GP with the AAV-GFAP-Cre-GFP (GFAP-D2-GP+/+, n12), and D2 flox/flox littermates bilaterally injected in the GPe with the AAV-GFAP-Cre-GFP (GFAP-D2-GP-/-, n14). GFAP-D2-GP-/- have increased PPI compared to GFAP- D2-GP+/+ mice (Two-way repeated measure ANOVA; genotype effect: F 1,25 =4.70 p=0.040). *p<0.05 vs GFAP-D2-GP+/+. c. Breakpoint during a food-driven operant behavior test with increasing progressive ratio (PR) displayed by GFAP-D2-GP+/+ (n12), and GFAP-D2-GP-/- (n12) littermates. GFAP-D2-GP-/- mice showed higher breakpoints than GFAP-D2-GP+/+ mice (Two-way repeated measure ANOVA; genotype effect: F 1,22 =5.78; p=0.025). *p<0.05 vs GFAP-D2-GP+/+. d. GFAP-D2-GP+/+ (n11) and GFAP-D2-GP-/- (n8) littermates were implanted with a dialysis probe for measurement of basal extracellular dopamine levels. Top panel show the localization of probe dialyzing portion within the GPe. Three mice were excluded for misplaced probe. GFAP-D2-GP-/- show higher extracellular dopamine levels compared to GFAP-D2-GP+/+ mice (t-test: t 14 =-2.13, p = 0.05). *p<0.05 vs GFAP-D2-GP+/+. e. Experimental design to selectively delete dopamine D2 receptors in the astrocytes of adult D2 flox/flox mice only within SNc/VTA, bilaterally. Example images of SNc/VTA stained for tyrosine hydroxylase (TH, to visualize the dopaminergic neurons) and for GFP (to identify the cells infected by the virus). f. Distribution of GFAP-positive astrocyte in the VTA/SN section co-stained for TH as a maker for the dopaminergic neurons. g-h. GFAP-D2- VTA/SN+/+ (n6) and GFAP-D2-SN/VTA-/- (n6) littermates were implanted with a dialysis probe for measurement of basal extracellular dopamine levels in the GPe (top figure shows the localization of probes dialyzing portion within the GPe). GFAP-D2-SN/VTA-/- showed increased basal extracellular g. dopamine (t-test: t 10 =-3.20, p = 0.009), and h. DOPAC levels (t-test: t 10 =-3.10, p = 0.011) than GFAP-D2-SN/VTA+/+ littermates. *p<0.05 vs GFAP-D2-SN/VTA+/+. i. Breakpoint during a food-driven operant behavior test with increasing progressive ratio (PR) displayed by GFAP-D2- SN/VTA+/+ (n10), and GFAP-D2-SN/VTA-/- (n14) littermates. GFAP-D2-SN/VTA-/- mice showed higher breakpoints than GFAP-D2-SN/VTA+/+ mice (Two-way repeated measure ANOVA; geno-type effect: F 1,22 =5.28; p=0.031). *p<0.05 vs GFAP-D2-SN/VTA+/+. j. Schematic figure model depicting astrocytes-dependent Dys1A/D2 signaling pathways involved in basal ganglia dopamine-related modulation of motivational and sensorimotor gating abilities.

Article Snippet: Subsequently they were incubated overnight at 4 °C with 1:300 rabbit polyclonal anti-GFAP antibody (Novus Biologicals, Centennial, CO, USA) in blocking solution.

Techniques: Injection, Control, Staining, Infection, Virus, Protein-Protein interactions

Journal: Biochimica et biophysica acta

Article Title: Thymosin β4 induces the expression of vascular endothelial growth factor (VEGF) in a hypoxia-inducible factor (HIF)-1α-dependent manner.

doi: 10.1016/j.bbamcr.2010.07.005

Figure Lengend Snippet: Fig. 4. Expression pattern of the thymosin β4, and HIF-1α in colon cancer. Tissue microarray slides were immunostained with rabbit polyclonal antibody to thymosin β4 (1: 500), and with mouse monoclonal antibody to HIF-1α (1:500 dilution). Slides treated with anti-thymosin β4 and HIF-1α were then incubated with fluorescence labeled secondary antibodies (Alexa Fluor 546 anti-mouse antibody and Alexa Fluor 488 anti-rabbit antibodies through the confocal laser-scanning microscope.

Article Snippet: Then slides were incubated with a mixture of rabbit polyclonal antibody to thymosin β4 (1: 500 dilution; ALPCODiagnostics,Windham,NH,USA), andmousemonoclonal antibody to HIF-1α (1:100 dilution; Novus Biologicals, Littleton, CO) at 4 °C overnight.

Techniques: Expressing, Microarray, Incubation, Labeling, Laser-Scanning Microscopy

Journal: Cancers

Article Title: A Systemic and Integrated Analysis of p63-Driven Regulatory Networks in Mouse Oral Squamous Cell Carcinoma.

doi: 10.3390/cancers15020446

Figure Lengend Snippet: Figure 11. Immunohistochemical staining in HNSCC tumor microarray tissues. Staining of p63 (A), COTL1 (B), and K14 (C) across normal, malignant tumor stage II, and malignant tumor stage III tissues at 10× magnification.

Article Snippet: After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. RoseAnne Romano) [29], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Trisbuffered saline with 0.05% Tween-20.

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: bioRxiv

Article Title: Selective reduction of KCNA4 in vulnerable glutamatergic-serotonin neurons of the dorsal raphe nucleus in Alzheimer’s Disease

doi: 10.1101/2025.10.17.683113

Figure Lengend Snippet: A–C: Confocal images demonstrating spatial distribution via RNAscope labeling for TPH2 ( A ), SLC17A8 (VGLUT3) ( B ) and 5HT/glut neurons ( C ) in human DRN; aq : aqueduct. Annotated box is expanded in ( D ). D: Immunofluorescence-enabled in situ hybridization (iFISH) labeling for MAP2 (microtubule-associated protein 2), TPH2 , SLC17A8 , and AT8 (phospho-tau antibody). Yellow arrows : 5HT/glut; white arrows : 5HT-nonglut. E: Micro-array data for subcortical brain areas from the ALLEN Human Brain Atlas for TPH2 , SLC17A8 , KCNA4 , and SLC24A5 (Case #H0351.2001). F: Percent immunoreactive area ( %IR ) for AT8 within all TPH2 -positive area (5-HT), TPH2 -positive / SLC17A8 -positive area (5HT/glut) and TPH2 -positive / SLC17A8 -negative (5HT-nonglut) area for all tau-positive human cases. Orange data points denote cognitively-normal clinical status. G: Percent expression area ( %EA ) for KCNA4 within 5-HT, 5HT/glut and 5HT-nonglut areas, respectively. Dataset includes all cases; lime data point denotes cognitively-normal PART case (tau-negative DRN). H: Same as ( G ), but for SLC24A5 . F–H: 1w RM ANOVA w/Tukey’s post hoc . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Slides were then incubated overnight at 4 °C with primary antibodies against MAP2 (chicken, #NB300-213, Novus Biologicals; 1:200) and phosphorylated tau (AT8, mouse, #MN1020, Thermo Fisher Scientific; 1:100).

Techniques: RNAscope, Labeling, Immunofluorescence, In Situ Hybridization, Microarray, Expressing

Journal: Cancers

Article Title: A Systemic and Integrated Analysis of p63-Driven Regulatory Networks in Mouse Oral Squamous Cell Carcinoma.

doi: 10.3390/cancers15020446

Figure Lengend Snippet: Figure 11. Immunohistochemical staining in HNSCC tumor microarray tissues. Staining of p63 (A), COTL1 (B), and K14 (C) across normal, malignant tumor stage II, and malignant tumor stage III tissues at 10× magnification.

Article Snippet: After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. RoseAnne Romano) [29], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Trisbuffered saline with 0.05% Tween-20.

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Viability of AML cell lines treated with Nut-3a and/or WIP1i. ( A ) Percentage of viable MOLM-13, MV-4-11, OCI-AML3, NOMO-1, HEL and KASUMI-1 AML cells treated with increasing concentrations of single agent Nut-3a (from 0.5 to 5 μM) for 24, 48 and 72 h. ( B ) IC50 values of AML cell lines at 72 h of treatment with Nut-3a or WIP1i (NR = not reached). ( C ) Percentage of viable cells treated with increasing concentrations of single agent WIP1i (from 5 to 20 μM) for 24, 48 and 72 h. Inhibition of cell viability induced in TP53 -wt ( D ) and TP53 -mut ( E ) AML cell lines by the combination of increasing concentrations of Nut-3a (from 0.5 to 5 μM) and WIP1i (from 5 to 20 µM) at 24, 48 and 72 h. Average value and standard deviation of 3 independent experiments are shown.

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Inhibition, Standard Deviation

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Apoptotic response of AML cell lines and primary cells to combined Nut-3a and WIP1i treatment. ( A ) Histograms showing the percentage of apoptotic (AnnexinV + cells) cells in TP53 -wt ( A ) and TP53 -mut cell lines ( B ) after 24 and 48 h treatment with single and combined Nut-3a and WIP1i. Average value and standard deviation of 3 independent experiments are shown. ( C ) Cell viability and ( D ) apoptotic response of TP53 -wt AML primary cells ( n = 3 and n = 6, respectively) after 24 h and 48 h treatment with single and combined Nut-3a and WIP1i treatments. ( E ) Histograms showing the percentage of dead cells in NPM1 -mut and NPM1 -wt primary AML cells ( n = 5 each) after 48 h treatment with single and combined Nut-3a and WIP1i. ( F ) Histograms showing the percentage of viable cells (normalized on vehicle-treated cells) in TP53 -mut ( n = 3) and TP53 -wt ( n = 4) primary AML cells after 48 h treatment with single and combined Nut-3a and WIP1i (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Standard Deviation

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Changes in the expression of p53-related genes induced by the treatment in TP53 -wt and TP53 -mut cells. Cells were harvested after 16 h of treatment (Nut-3a 0.5 and 5 µM; WIP1i 5 and 20 µM, for TP53 -wt and TP53 -mut cells, respectively) both for gene expression microarray and protein analyses. ( A ) Enrichment of p53 signature in MV-4-11 cells treated with the drug combination vs. vehicle (gene expression microarray). ( B ) Heatmap of MV-4-11 and NOMO-1 cells showing the significantly deregulated genes (differential expression analysis between Nut-3a+WIP1i-treated and vehicle-treated MV-4-11 cells, fold change ≥ 2, p < 0.05) belonging to the p53 signature in the analyzed models. ( C ) Protein quantification of p53-related genes in treated TP53 -wt and ( D ) TP53 -mut cells. Histograms show the average value of 3 independent experiments ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Expressing, Microarray

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Most significant enriched pathways in MV-4-11 cells upon drug treatment.

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Comparison, Activity Assay

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Proposed mechanism of action of Nut-3a and WIP1i combined treatment in AML cells. MDM2 inhibition enhances p53-dependent response to DNA damages induced by chemotherapy agents or replicative stress. Once Nut-3a binds to MDM2, p53 is released and activated through phosphorylation. Active p53 promotes the induction of apoptosis. ( A ) WIP1 is involved in the regulation of response to Nut-3a and dephosphorylates p53. WIP1 and p53 are co-regulated by a feedback-loop. ( B ) When WIP1i is simultaneously added to Nut-3a, p53 activation is enforced, resulting in enhanced apoptosis of AML cells. The arrows represent a stimulatory signal, truncated arrows represent a inhibition signal.

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Inhibition, Activation Assay

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 1. MUCL1 gene expression analysis in CRC. (A) MUCL1 expression in CRC (fold‑change) compared with adjacent normal tissue based on microarray data. Data are presented as the mean ± SD. (n=13). (B) Boxplot comparing the expression of MUCL1 in a cohort of COAD (n=275) compared with normal colon tissue (n=349) from the TCGA and GTEx datasets. (C) Boxplot comparing the MUCL1 expression in a cohort of READ (n=92) compared with normal rectal tissue (n=318) from the TCGA and GTEx datasets. (D) Stage plot of MUCL1 expression in colon cancer stages in COAD. (E) Stage plot showing MUCL1 expression in rectal cancer stages in READ. *P<0.05 vs. normal. MUCL1, mucin‑like 1; CRC, colorectal cancer; COAD, colon adenocarcinoma; READ, rectal adenocarcinoma; TCGA, The Cancer Genome Atlas; GTEx, Genotype Tissue Expression.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Gene Expression, Expressing, Microarray

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 2. MUCL1 transcription expression in various clinicopathological parameters of COAD. (A) Primary tumors and cancer stages. (B) Histological subtype and nodal metastasis. (C) Sex and race based. (D) Weight and age group based and (E) Based on TP‑53 mutation status. *P<0.05, **P<0.01 vs. normal. MUCL1, mucin‑like 1; COAD, colon adenocarcinoma; TCGA, The Cancer Genome Atlas.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 3. MUCL1 protein expression in human CRC cell lines. (A) Soluble protein of whole cell lysate from HT‑29, SW480 and SW620 cells was immunob‑ lotted against the indicated antibodies. (B) Total cell lysate from HT‑29‑Control siRNA and HT‑29‑MUCL1siRNA clones ML1 and 2 was immunoblotted against the indicated antibodies. (C) Total cell lysate from SW620 Control siRNA and SW620‑MUCL1siRNA clones ML1 and 2 was immunoblotted against the indicated antibodies. (D) Total cell lysate from HT‑29‑Control siRNA and HT‑29‑MUCL1 siRNA clones ML1 and 2 was evaluated for the expression of Bcl2, BclxL and caspase‑3. (E) Total cell lysate from SW620‑Control siRNA and SW620‑MUCL1siRNA clones ML1 and 2 was immunoblotted against the indicated antibodies (Bcl2, BclxL and caspase‑3). Densitometric analysis was conducted as follows: Intensity of protein bands were semi‑quantified and plotted as relative protein expression to control. The bar graphs are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control. MUCL1, mucin‑like 1; CRC, colorectal cancer; si‑, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Clone Assay, Control

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 4. MUCL1 promotes cell proliferation. (A) HT‑29‑Control siRNA and HT‑29‑MUCL1 siRNA clones ML1 and 2 and (B) SW620‑Control siRNA and SW620‑MUCL1 siRNA clones ML1 and 2 cells were seeded (500/well) in 6‑well plate and incubated at 37˚C. After 10‑12 days of incubation crystal violet staining was performed, colonies were quantified and images were captured by Bio‑Rad gel‑doc system. (C) HT‑29‑Control siRNA and HT‑29‑MUCL1 siRNA clones ML1 and 2 were seeded at 5,000 cells/well in 96‑well plates and incubated at 37˚C. Cell proliferation was determined by Cell Counting Kit‑8 assay on 24, 48, 72 and 96 h. (D) SW620‑Control siRNA and SW620‑MUCL1siRNA clone ML1 and 2 cells were seeded at 5,000 cells/well in 96‑well plate for incubation at 37˚C. Cell proliferation was measured by CCK‑8 assay on 24, 48, 72 and 96 h. The results are expressed as the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control. MUCL1, mucin‑like 1; si‑, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Clone Assay, Incubation, Staining, CCK-8 Assay, Control

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 5. Targeting MUCL1 inhibits migration and invasion. Transwell migration and invasion assay was performed using 24‑well plates. Migration and invasion activity were evaluated after 48 h by crystal violet staining. Quantification of migration was measured as relative migration and compared with control. (A) Migration ability of HT‑29‑Con siRNA and HT‑29‑MUCL1siRNA clones ML1 and 2. (B) Migration ability of SW620‑Con siRNA and SW620‑MUCL1siRNA clones ML1 and 2 plated in Transwell plate inserts. (C) Invasion ability of HT‑29‑Con siRNA and HT‑29‑MUCL1siRNA clones ML1 and 2. (D) Invasion ability of SW620‑Con siRNA and SW620‑MUCL1siRNA clones ML1 and 2 plated in Transwell plate inserts. Invasive activity was determined as relative invasion. All assays were carried out in triplicate. Scale bar, 100 µm. The results were expressed as the mean ± SD of three independent experiments. ***P<0.001 vs. control. MUCL1, mucin‑like 1; si‑, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Migration, Invasion Assay, Activity Assay, Staining, Control, Clone Assay

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 6. MUCL1 induces epithelial‑mesenchymal transition by activating β‑catenin. Total cell lysates from (A) HT‑29‑Control siRNA and HT‑29‑MUCL1 siRNA clones ML1 and 2 or (B) from SW620‑Control siRNA and SW620‑MUCL1 siRNA clones ML1 and 2 cells were immunoblotted against the indi‑ cated antibodies (E‑cadherin and vimentin). Total cell lysates from (C) HT‑29‑Control siRNA and HT‑29‑MUCL1 siRNA clones ML1 and 2 or (D) from SW620‑Control siRNA and SW620‑MUCL1 siRNA clones ML1 and 2 cells were immunoblotted against the indicated antibodies (phosphor‑β‑catenin‑Ser‑552 and β‑catenin). Nuclear and cytosolic extracts from (E) HT‑29‑Control siRNA and HT‑29‑MUCL1 siRNA clones ML1 and 2 or (F) from SW620‑Control siRNA and SW620‑MUCL1 siRNA clones ML1 and 2 were immunoblotted against the indicated antibodies (β‑catenin, β‑actin and Lamin B). Densitometric analysis was conducted as follows: Intensity of the protein bands was semi‑quantified and plotted as relative protein expression to control. The bar graphs are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control. MUCL1, mucin‑like 1; si‑, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Clone Assay, Expressing, Control

Journal: International journal of oncology

Article Title: Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

doi: 10.3892/ijo.2022.5312

Figure Lengend Snippet: Figure 7. Targeting MUCL1 increases sensitivity towards IRI. (A) HT‑29‑Control siRNA and HT‑29‑MUCL1siRNA clones ML1 and 2 cells were treated with IRI (50 and 100 µM) for 24 h. (B) Percentage of total cell death was analyzed by Annexin V/PI staining using flow cytometry. (C) SW620‑Control siRNA and SW620‑MUCL1siRNA clones ML1 and 2 cells were exposed to IRI (50 and 100 µM) for 24 h. (D) Total cell death (%) was analyzed by Annexin V/PI staining using flow cytometry. Total cell death (%) shown is representative of three independent experiments (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. control. HT‑ML1/HT‑ML2 vs. control (black); HT‑ML1‑IRI‑50/HT‑ML2‑IRI‑50 vs. HT‑IRI‑50 (blue). SW‑ML1/SW‑ML2 vs. control (black); SW‑ML1‑IRI‑50/SW‑ML2‑IRI‑50 vs. SW‑IRI‑50 (blue); SW‑ML1‑IRI‑100/SW‑ML2‑IRI‑100 vs. SW‑IRI‑100 (red). MUCL1, mucin‑like 1; si‑, small interfering; Con, control.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 ̊C with the following primary antibodies: MUCL1 (cat. no. NBP1‐92366; 1:500; Novus Biologicals, LLC), Bcl2 (cat. no. sc‐492; 1:1,000), BclxL (cat. no. sc‐56021; 1:1,000), caspase‐3 (cat. no. sc‐56053; 1:1,000), E‐cadherin (cat. no. sc‐8426; 1:1,000), vimentin (cat. no. sc‐6260; 1:1,000), Lamin B (cat. no. sc‐374015; 1:1,000) and β‐actin (cat. no. sc‐47778; 1:2,000) (all from Santa Cruz Biotechnology, Inc.).

Techniques: Clone Assay, Staining, Flow Cytometry, Control

Journal: Frontiers in Endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: GCM1 is expressed in equine trophoblast and expression correlates with LHB . (A) mRNA expression of LHB in ChG and chorion tissues at days 27, 30, 31, and 34 of pregnancy. (B) mRNA expression of GCM1 in ChG and chorion tissues at days 27, 30, 31, and 34 of pregnancy. (C) Correlation of mRNA expression of LHB and GCM1 in individual ChG tissues. *** p < 0.001, **** p < 0.0001 relative to day 27 ChG, ### p < 0.001, #### p < 0.0001 relative to CH at same time point (linear mixed-effects modeling, SPSS). (D) Glial cells missing 1 (GCM1) protein expression in untransfected COS7 cells and COS7 cells transfected with 250 ng of equine pCMV-myc-GCM1 expression vector. B-Actin expression was used as a loading control. (E) GCM1 and B-Actin protein expression in primary equine day 34 conceptus tissues: chorionic girdle (ChG), chorion (CH), allantochorion (ALC), and yolk sac (YS).

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Expressing, Transfection, Plasmid Preparation, Control

Journal: Frontiers in Endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: Glial cells missing 1 (GCM1) transactivates the LHB promoter. (A) Western blotting of protein extracted from human choriocarcinoma BeWo cells ( n = 3) using an antihuman GCM1 monoclonal antibody (c-terminal). β-actin was used as a loading control. (B) BeWo cells were transfected with specific lengths of the LHB promoter in a pGL3-basic luciferase reporter vector ( n = 3). Activity of promoter constructs was expressed as fold-change firefly/renilla compared to pGL3-basic. ** p < 0.01, *** p < 0.001 (one-way ANOVA). (C) pGL3-basic and pGL3-335 were co-transfected with ratios of pCMV-myc-Empty: pCMV-myc-GCM1 to assess ability of GCM1 to drive promoter activity in COS7 cells ( n = 3). * p < 0.05, *** p < 0.001, **** p < 0.0001 relative to pGL3-335 alone, ## p < 0.01 relative to pGL3-335 + 250 ng pCMV-myc-Empty, ˆˆˆ p < 0.001 relative to pGGl-335 + 150 ng pCMV-myc-GCM1 (two-way ANOVA). (D) Specific lengths of the LHB promoter were transfected with 150 ng of empty pCMV-myc-Empty vector, or with 150 ng of pCMV-myc-GCM1 ( n = 4). * p < 0.05, ** p < 0.0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA).

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Western Blot, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct

Journal: Frontiers in Endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: Glial cells missing 1 (GCM1) binds to the proximal LHB promoter in primary equine chorionic girdle (ChG) trophoblast cells and mutation of GCM1 site 1 results in loss of promoter activity. (A) Chromatin ImmunoPrecipitation was carried out on formaldehyde fixed chromatin complexes from passage 1 day 34 primary ChG trophoblasts, using an antihuman GCM1 antibody for immunoprecipitation. Binding of GCM1 to predicted GCM1-binding sites was expressed as fold-change enrichment over binding to a control region within the coding sequence of the LHB gene ( n = 2 conceptuses). (B,C) The two GCM1-binding sites in the pGL3-335 promoter construct were mutated. Red boxes depict GCM1 binding sites. Red box with a cross depicts a mutated site. Wild-type pGL3-335 and its two mutant constructs, pGL3-335-mut1 and pGL3-335-mut2, were co-transfected (B) into BeWo cells ( n = 3) (C) into COS7 cells, with 150 ng of pCMV-myc-GCM1 or pCMV-myc-Empty as a control ( n = 4). Promoter activity was assessed by luciferase assay. * p < 0.05, ** p < 0.01, **** p < 0.0001 [ (B) one-way ANOVA, (C) two-way ANOVA].

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Mutagenesis, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Control, Sequencing, Construct, Transfection, Luciferase

Journal: Frontiers in Endocrinology

Article Title: Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter

doi: 10.3389/fendo.2018.00195

Figure Lengend Snippet: Glial cells missing 1 (GCM1) binding partners are differentially expression in vivo during differentiation of chorionic girdle (ChG) trophoblast cells. (A) mRNA expression of ETV1, ETV7, HOXA13 , and PITX1 in ChG and chorion tissues between days 27 and 34 of pregnancy, as determined by microarray analysis ( n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 relative to day 27 ChG, # p < 0.05, ## p < 0.01, ### p < 0.001, relative to time matched chorion. (B) Schematic of the LHB promoter showing directly bound GCM1-binding site and region of potential binding by other transcription factors. TLSS is translational start site, TSS shown by black arrow is transcriptional start site. Numbers are relative to TLSS.

Article Snippet: Following activation in methanol and blocking for 1 h in Tris-buffered saline-Tween 20 (TBS-T) containing 5% (wt/vol) nonfat milk, membranes were incubated overnight at 4°C in a 1:1,000 dilution of rabbit anti-human GCM1 polyclonal antibody (Aviva Systems Biology), in TBS-T containing 5% (wt/vol) nonfat milk.

Techniques: Binding Assay, Expressing, In Vivo, Microarray