Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants reduces CTCs. MDA-MB-231 cells with constitutive sh mdm2 , sh mdmx , or mir30 shRNA-expressing vector were implanted into the mammary fat pads of 6-week-old female NSG mice. a Western blot analysis of MDM2, MDMX, and mtp53 protein levels from 50 μg of whole-cell lysates from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively) prior to mammary fat pad implantation. Actin is shown as a loading control. b Box-and-whisker plot represents the numbers of CTCs per milliliter from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells engrafted into animals. The number of CTCs was determined by flow cytometry, and the total events were counted (gates were set by the GFP signal intensity and cell size). The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals. The adjusted p value was obtained with two-tailed, two-sample t tests using a permutation test. c Representative fluorescence-activated cell sorting plots showing GFP-positive events in different mouse groups

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Plasmid Preparation, Western Blot, Control, Whisker Assay, Flow Cytometry, Two Tailed Test, Fluorescence, FACS

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants does not significantly reduce primary tumor growth. a Primary tumor volumes of 231.mir30.vector ( n = 6), 231.sh mdm2 ( n = 7), and 231.sh mdmx (n = 7) engrafted animals were measured using calipers over 36 days. b The endpoint tumor volumes were determined on dissected masses at the time of necropsy. c mRNA levels of mdm2 and mdmx normalized to gapdh in primary tumors were determined by real-time qRT-PCR. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = nonsignificant. The p value was calculated using two-tailed unpaired t tests. d Protein expression of MDM2, MDMX, and mtp53 from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx engrafted primary tumors were determined by Western blot analysis. Three tumors per group were used, and actin is shown as a loading control

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Control

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 provoke in vitro MDA-MB-231 cell migration without altering cell proliferation. a Representative Western blot demonstrating the levels of MDM2, MDMX, and mtp53 in 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively). Fifty micrograms of lysate was loaded per lane. Actin was used as a loading control. b The number of cells was determined by hemocytometer cell counting. Cells ( n = 50,000) were seeded in triplicate, and cell counting was performed at 2, 4, 5, and 6 days. Dots represent mean values, and error bars represent SD. Experiments were carried out with three biological replicates. c Wound closure was observed by phase-contrast microscopy and photographed at 0 and 12 h. One representative image from each group at 0 and 12 h is shown. d The wound area was measured by using NIS-Elements software (Nikon Instruments, Melville, NY, USA). The percentage of wound closure was quantified from four independent biological experiments. The p value was obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: In Vitro, Migration, Western Blot, Plasmid Preparation, Control, Cell Counting, Microscopy, Software, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX knockdown in primary tumors blocks the transcription of CXCR4 and PTGS2 . The 231.mir30.vector-, 231.sh mdm2 -, and 231.sh mdmx -derived primary tumors were lysed and used for total RNA extraction and complementary DNA synthesis. a Microarray analysis revealed selected tumor metastasis-related genes that were either up- or downregulated in 231.sh mdm2 and 231.sh mdmx compared with 231.mir30 vector. Fold changes were gated either > 2 or < 0.5. Two tumor samples per group were used for the analysis. b From the respective cells derived from all the primary tumors, the total CXCR4 and PTGS2 levels were determined by real-time qRT-PCR, and these were compared with those of the parental cells grown in culture. The bars represent mean values, and error bars represent SD. The p values were obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Derivative Assay, RNA Extraction, DNA Synthesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDM2 knockdown in ERα + T47D orthotopic transplant reduces tumor volume. a T47D cells with inducible sh mdm2 or mir30 shRNA-expressing control vector were treated with 4 μg/ml doxycycline (Dox) for 10 days to induce and maintain shRNA expression. Western blot shows the levels of MDM2, MDMX, E-cadherin, and mtp53 with Dox treatment (lanes 1 and 2) prior to mammary fat pat implantation. Actin was used as a loading control. b Animals were provided with 2 mg/ml Dox and 8 μg/ml E 2 in their drinking water during the entire experiment. Primary tumor growth was measured over a period of 60 days using calipers *** p < 0.001 calculated by two-tailed unpaired t test. c The experimental endpoint tumor volume was determined at the time of necropsy. d mdm2 mRNA expression in primary tumors was determined by real-time qRT-PCR. The p value was determined by two-tailed unpaired t test. e E-cadherin, MDM2, MDMX, and mtp53 protein levels from primary tumors were determined by Western blot analysis. Actin was used as the loading control. f Representative H&E staining images of T47D.vector and T47D.sh mdm2 under 200× and 1000× magnification. T represents Tumor; nm represents normal mammary fat pad; M represents muscle; arrowhead depicts tumor cells infiltrating into muscle layer

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Control, Plasmid Preparation, Western Blot, Two Tailed Test, Quantitative RT-PCR, Staining

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: Comparative levels of CXCR4 in ERα + T47D and TNBC MDA-MB-231 tumors. a CXCR4 RNA expression normalized to gapdh with MDM2 knockdown was determined using real-time qRT-PCR. 231.mir30.vector ( n = 6), T47D.mir30.vector ( n = 10), and T47D.sh mdm2 ( n = 9) tumor samples were analyzed. The RNA level of CXCR4 was set as 1 for the 231.mir30.vector group, and T47D samples were expressed relative to 231.mir30.vector values. b Protein expression of CXCR4 was compared from parental cell lines and two tumors from each group. Representative Western blot demonstrating protein levels of CXCR4 and actin in MDA-MB-231 and T47D groups shown using two gels (lanes 1–9 and 10–16, with tumor 2 for 231.sh mdmx used in lane 10 as a common reference)

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: RNA Expression, Knockdown, Quantitative RT-PCR, Plasmid Preparation, Expressing, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 in TNBC promote metastasis, and in ERα + breast cancer MDM2 promotes proliferation. In TNBC, MDMX promotes expression of CXCR4 and PTGS2 with associated release of CTCs but no increase in cell proliferation. In ERα + breast cancer, estrogen stimulates MDM2 expression with no influence on CXCR4 and causes an increase in cell proliferation without correlated metastasis

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Expressing

Journal: Nature

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate

doi: 10.1038/nature15721

Figure Lengend Snippet: a , Histogram of frequency of genes for different levels of expression enrichment in carotid body (CB) relative to adrenal medulla (AM) by RNA sequencing. Log 2 (CB/AM) values are shown, with data binned for every log 2 interval of 1.0 centered at integers. b , Plot of log 2 values of reads per kilobase per million (RPKM) in CB and AM of all 1,126 olfactory receptor (OR) genes annotated in RefSeq shown in alphanumerical order. The five OR genes expressed at RPKM > 2 (dashed line) are indicated. Samples that had no transcripts are plotted at a value of −7.1, just below the smallest RPKM value for ORs. Data presented in . c , Comparison of expression levels of >34,000 genes in adult mouse CB and AM by whole genome microarrays. Plot shows log 2 of the ratio for CB relative to AM of the fluorescence intensity values for the 45,000 probe sets. The three probe sets for Olfr78 transcripts are indicated (circles). Expression of Olfr78 was significantly different between CB and AM for all three probe sets ( P <0.05 by ANOVA with false discovery rate control). d , Histogram of the frequency of genes for different levels of expression enrichment in CB relative to AM in microarray data. Log 2 (CB/AM) values are shown, with data binned for every log 2 interval of 1.0 centered at integers. The three probe sets detecting Olfr78 mRNA (arrows) confirmed the RNA-seq data ( a , , and ) showing Olfr78 among the mRNAs most highly enriched in carotid body. Mouse CB Olfr78 expression is consistent with previous microarray data , . a-d , n =3 cohorts of 10 animals each. Data as mean. e , Genomic locus showing the large cluster of ~160 Class I OR genes on chromosome 7, with region encoding MOR18 subfamily ( Olfr78 , Olfr558 , and Olfr557 ) expanded below. We did not detect transcripts in either tissue for Olfr557 , which lies adjacent to Olfr558 in the cluster, or for the intervening ( Olfr33 , Olfr559 ) and intronic ( Olfr560 ) ORs. Clusters of genes encoding globins, Trims, and USP proteins are also found with this OR cluster. Large box, coding sequence; arrowhead, coding orientation; small box, non-coding exons.

Article Snippet: Other mouse strains used were: Olfr78 knock-in reporter: MOL2.3- IGITL , kindly shared by Ron Yu (Stowers Institute) Olfr78 knock-in mutant/reporter: B6; 129P2- Olfr78 tm1Mom /MomJ, Stock #006722 (JAX) Olfr558 mutant/reporter: B6129S5- Olfr558 tm1Lex , Stock #TF0586 (Taconic) Th-Cre driver: B6.FVB(Cg)- Tg(Th-cre) FI172Gsat /Mmucd, Stock #031029-UCD (MMRRC) Th-Cre driver: Th tm1(cre)Te , kindly shared by Karl Deisseroth (Stanford) ROSA-GCaMP3: B6;129S- Gt(ROSA)26Sor tm38(CAG-GCaMP3)Hze /J, Stock #014538 (JAX) ROSA-tdTomato: B6;129S6- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J, Stock #007905 (JAX) Adult animals were used in all experiments, unless indicated otherwise.

Techniques: Expressing, RNA Sequencing, Comparison, Fluorescence, Control, Microarray, Sequencing

Journal: Nature

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate

doi: 10.1038/nature15721

Figure Lengend Snippet: Expression of Olfr78 reporter in heterozygous ( a ) and homozygous ( b-d ) Olfr78-GFP-taulacZ reporter animals . a-c , Sections of carotid bifurcations stained for GFP ( Olfr78 reporter; green), tyrosine hydroxylase (TH; red), and DAPI (nuclei; blue). a , Section of CB showing co-expression of reporter GFP and TH in glomus cells. Monoallelic expression would predict that only half of TH-positive cells express the reporter . Arrowheads, clusters of glomus cells expressing both GFP and TH. b, c , Sections of the same carotid bifurcation. Panels on right show close-ups of boxed region (petrosal ganglion, PG). No GFP-positive cells were found in petrosal ganglion. TH-positive nerve fibers (arrowheads) and cell bodies were found in glossopharyngeal nerve (GN) and petrosal ganglion. Dashed circle indicates vagus nerve (VN). NG/JG, nodose/jugular ganglia. d , X-gal staining of a brain sagittal section. Reporter expression (blue) was restricted to olfactory bulb (arrowhead) in this section and complete brain serial sagittal sections. Anterior, right; dorsal, up. e-h , Olfr558 expression in a knockout/reporter mouse in which the Olfr558 coding region is replaced with lacZ encoding β-galactosidase. e , Olfr558 reporter expression in blood vessels of CB and SCG by X-gal staining. Heterozygous Olfr558 +/lacZ samples showed the same pattern of staining (data not shown). f-h , CB sections immunostained for β-galactosidase ( Olfr558 reporter; green), TH (red), with DAPI counterstain (blue) in f , and for β-galactosidase (green) and CD31 (red) in g or smooth muscle actin (red) in h . Scale bars, 100 µm ( a, b-right, c-right, f-h ), 200 µm ( b-left, c-left ), 500 µm ( e ), and 2 mm ( d ).

Article Snippet: Other mouse strains used were: Olfr78 knock-in reporter: MOL2.3- IGITL , kindly shared by Ron Yu (Stowers Institute) Olfr78 knock-in mutant/reporter: B6; 129P2- Olfr78 tm1Mom /MomJ, Stock #006722 (JAX) Olfr558 mutant/reporter: B6129S5- Olfr558 tm1Lex , Stock #TF0586 (Taconic) Th-Cre driver: B6.FVB(Cg)- Tg(Th-cre) FI172Gsat /Mmucd, Stock #031029-UCD (MMRRC) Th-Cre driver: Th tm1(cre)Te , kindly shared by Karl Deisseroth (Stanford) ROSA-GCaMP3: B6;129S- Gt(ROSA)26Sor tm38(CAG-GCaMP3)Hze /J, Stock #014538 (JAX) ROSA-tdTomato: B6;129S6- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J, Stock #007905 (JAX) Adult animals were used in all experiments, unless indicated otherwise.

Techniques: Expressing, Staining, Knock-Out

Journal: Nature

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate

doi: 10.1038/nature15721

Figure Lengend Snippet: Top 150 genes highly expressed in carotid body vs. adrenal medulla by RNA-seq.

Article Snippet: Other mouse strains used were: Olfr78 knock-in reporter: MOL2.3- IGITL , kindly shared by Ron Yu (Stowers Institute) Olfr78 knock-in mutant/reporter: B6; 129P2- Olfr78 tm1Mom /MomJ, Stock #006722 (JAX) Olfr558 mutant/reporter: B6129S5- Olfr558 tm1Lex , Stock #TF0586 (Taconic) Th-Cre driver: B6.FVB(Cg)- Tg(Th-cre) FI172Gsat /Mmucd, Stock #031029-UCD (MMRRC) Th-Cre driver: Th tm1(cre)Te , kindly shared by Karl Deisseroth (Stanford) ROSA-GCaMP3: B6;129S- Gt(ROSA)26Sor tm38(CAG-GCaMP3)Hze /J, Stock #014538 (JAX) ROSA-tdTomato: B6;129S6- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J, Stock #007905 (JAX) Adult animals were used in all experiments, unless indicated otherwise.

Techniques: Membrane, Binding Assay, Derivative Assay, Control

Journal: Nature

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate

doi: 10.1038/nature15721

Figure Lengend Snippet: Expression of genes associated with olfactory neurons.

Article Snippet: Other mouse strains used were: Olfr78 knock-in reporter: MOL2.3- IGITL , kindly shared by Ron Yu (Stowers Institute) Olfr78 knock-in mutant/reporter: B6; 129P2- Olfr78 tm1Mom /MomJ, Stock #006722 (JAX) Olfr558 mutant/reporter: B6129S5- Olfr558 tm1Lex , Stock #TF0586 (Taconic) Th-Cre driver: B6.FVB(Cg)- Tg(Th-cre) FI172Gsat /Mmucd, Stock #031029-UCD (MMRRC) Th-Cre driver: Th tm1(cre)Te , kindly shared by Karl Deisseroth (Stanford) ROSA-GCaMP3: B6;129S- Gt(ROSA)26Sor tm38(CAG-GCaMP3)Hze /J, Stock #014538 (JAX) ROSA-tdTomato: B6;129S6- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J, Stock #007905 (JAX) Adult animals were used in all experiments, unless indicated otherwise.

Techniques: Expressing, Marker

Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Representative images of the grading system used in the tissue microarray analysis. (A-E) Different staining intensities of PARK2 in (A) non-malignant and (B-E) ccRCC tissue are shown; (B) negative, (C) weakly positive, (D) moderately positive, (E) strongly positive. (F-I) Different staining intensities of CKS2 in (F) non-malignant (G-I) and ccRCC tissue (G) weakly positive, (H) moderately positive, (I) strongly positive.

Article Snippet: PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest Eco RI (Thermo Fisher Scientific, Inc.) and FastDigest Bam HI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).

Techniques: Microarray, Staining

Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: PARK2 mRNA is downregulated in ccRCC tissues and primary cells compared with non-malignant tissue. (A) PARK2 mRNA expression was examined by RT-qPCR, which showed lower PARK2 expression in tumor tissue of patients with ccRCC compared with non-malignant tissue. Data were analyzed using Wilcoxon signed-rank test; n=63; **** P<0.0001. (B) PARK2 mRNA expression levels in ccRCC tissue and primary cells compared with non-malignant tissue was measured by RT-qPCR. ccRCC tissue showed decreased PARK2 mRNA expression levels compared with non-malignant tissue, which were reduced even more when cells were cultivated. Data are presented as the mean ± SEM; n=7. ccRCC, clear cell renal cell carcinoma; PARK2, parkin; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest Eco RI (Thermo Fisher Scientific, Inc.) and FastDigest Bam HI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Generation and functional analyses of cell lines. (A) Transduction of ccRCC cell lines with a lentiviral vector with or without PARK2. (B) PARK2 mRNA expression was examined by reverse transcription-quantitative PCR, which showed higher mRNA expression levels in PARK2-transduced cells. TATA-box binding protein and β-actin were used as housekeeping genes. Kruskal-Wallis test was performed followed by Dunn's post hoc test; * P<0.05. (C) PARK2 protein expression levels were assessed by immunoblotting of the different transduced cell lines. β-actin was used as a loading control. Adjusted densities were measured in GraphPad Prism version 8.2.0. (D) Transwell Boyden chamber migration assays were performed, which demonstrated a difference in migrative capacity after 6-8 h incubation. Magnification, ×10. Data were compared by Kruskal-Wallis followed by Dun's; n=3; **** P<0.0001. (E) PARK2 overexpression reduced the invasiveness compared with control cells after 48 h incubation. Magnification, ×10. Kruskal-Wallis test was performed followed by Dunn's post hoc test; n=3; *** P<0.001, **** P<0.0001. Data are presented as the mean ± SEM of triplicate experiments. EF1a, elongation factor 1α; EV, empty vector; IRES, internal ribosomal entry site; PARK2, parkin; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type.

Article Snippet: PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest Eco RI (Thermo Fisher Scientific, Inc.) and FastDigest Bam HI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).

Techniques: Functional Assay, Transduction, Plasmid Preparation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot, Control, Migration, Incubation, Over Expression, Quantitative RT-PCR

Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: CKS2 protein levels are reduced in cells overexpressing PARK2. (A) Volcano plots depicting differential protein expression in the different transduced cells. Only slight differences in protein levels were observed for 786-O WT and 786-O EV cells (|log 2 FC|>1; count, 4), bigger differences were observed when comparing 786-O [|log 2 FC|>1; count, 43 (2 with FDR <0.05)] or 786-O EV cells [|log 2 FC|>1; count, 38 (2 with FDR <0.05)] with 786-O PARK2 cells. (B) Profile plots illustrated that the abundance of PARK2 peptides were increased in 786-O PARK2 cells. (C) Abundance of CKS2 peptides was decreased in 786-O PARK2 cells. CKS2, CDC28 protein kinase regulatory subunit 2; EV, empty vector; FC, fold change; PARK2, parkin; WT, wild-type.

Article Snippet: PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest Eco RI (Thermo Fisher Scientific, Inc.) and FastDigest Bam HI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).

Techniques: Expressing, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: An inactive catalytic domain mutant 786-O C431S exhibits a similar phenotype as the control cells. (A) Cells with a PARK2 C431S mutation were generated by transducing cells with the pLVX-EF1a-PARKC431S-IRES-mCherry plasmid. (B) PARK2 mRNA expression levels were examined by RT-qPCR. TBP and β-actin were used as housekeeping genes. Data are presented as the mean + SEM of triplicate experiments. Kruskal-Wallis test was performed followed by Dunn's post hoc test; * P<0.05. (C) RT-qPCR analysis revealed no difference in CKS2 mRNA expression levels between the different transduced ccRCC cell lines. Data are presented as the mean ± SEM. (D) Transwell Boyden chamber migration assays were conducted with mutant cell lines 786-O C431S and RCC-MH C431S . Magnification, ×10. Data were compared by Kruskal-Wallis followed by Dunn's post hoc test; n=3; * P<0.05, *** P<0.001, **** P<0.0001. (E) CKS2 mRNA expression levels were assessed after siRNA knockdown. The experiment was conducted in triplicate. TBP and β-actin served as housekeeping genes. Data are presented at the mean ± SEM. (F) Migratory capacity was assessed after siRNA-mediated knockdown of CKS2 and (G) significance was assessed by Kruskal-Wallis followed by Dunn's post hoc test; n=2; ** P<0.01, *** P<0.001. Magnification, ×10. ccRCC, clear cell renal cell carcinoma; CKS2, CDC28 protein kinase regulatory subunit 2; EF1a, elongation factor 1α; EV, empty vector; IBR, in-between RING fingers domain; IRES, internal ribosome entry site; ns, not significant; PARK2, parkin; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; TBP, TATA-box binding protein; Ubl, ubiquitin-like domain; WT, wild-type.

Article Snippet: PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest Eco RI (Thermo Fisher Scientific, Inc.) and FastDigest Bam HI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).

Techniques: Mutagenesis, Control, Generated, Plasmid Preparation, Expressing, Quantitative RT-PCR, Migration, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Binding Assay, Ubiquitin Proteomics

Journal: International Journal of Oncology

Article Title: Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

doi: 10.3892/ijo.2022.5310

Figure Lengend Snippet: Elevated CKS2 levels are associated with poor survival in patients with ccRCC. (A) Kaplan-Meier curves of the survival probability showing an improved overall survival for patients with low CKS2 expression levels compared with high CKS2 levels. Significance was assessed with log-rank test. Number at risk indicates the number of patients which are still alive at given time point; n=247. (B) Kaplan-Meier curves followed by log-rank test showing no significant difference in overall survival for patients with high PARK2 expression levels compared with low PARK2 levels; n=250. (C) Cox proportional hazard ratio showing an association between survival and different parameters, such as CKS2 expression, PARK2 expression, tumor grade and pT. * P<0.05, *** P<0.001 (D) Pearson's correlation analysis depicts a relationship between PARK2 and pT. In addition, CKS2 and pT were also analyzed. Bold values indicate significant p-values. (E) Spearman's correlation of CKS2 and grading as well as PARK2 and grading are depicted. (F) Pearson's correlation between CKS2 and PARK2 including Pearson's r, P-value, 95% CI, t-stat and df. ccRCC, clear cell renal cell carcinoma; CI, confidence interval; CKS2, CDC28 protein kinase regulatory subunit 2; df, degrees of freedom; PARK2, parkin; pT, pathologic tumor stage; t-stat, t-statistic.

Article Snippet: PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest Eco RI (Thermo Fisher Scientific, Inc.) and FastDigest Bam HI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants reduces CTCs. MDA-MB-231 cells with constitutive sh mdm2 , sh mdmx , or mir30 shRNA-expressing vector were implanted into the mammary fat pads of 6-week-old female NSG mice. a Western blot analysis of MDM2, MDMX, and mtp53 protein levels from 50 μg of whole-cell lysates from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively) prior to mammary fat pad implantation. Actin is shown as a loading control. b Box-and-whisker plot represents the numbers of CTCs per milliliter from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells engrafted into animals. The number of CTCs was determined by flow cytometry, and the total events were counted (gates were set by the GFP signal intensity and cell size). The number of CTCs per milliliter was obtained by dividing the number of positive events by blood volume from individual animals. The adjusted p value was obtained with two-tailed, two-sample t tests using a permutation test. c Representative fluorescence-activated cell sorting plots showing GFP-positive events in different mouse groups

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Plasmid Preparation, Western Blot, Control, Whisker Assay, Flow Cytometry, Two Tailed Test, Fluorescence, FACS

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 knockdown in MDA-MB-231 orthotopic transplants does not significantly reduce primary tumor growth. a Primary tumor volumes of 231.mir30.vector ( n = 6), 231.sh mdm2 ( n = 7), and 231.sh mdmx (n = 7) engrafted animals were measured using calipers over 36 days. b The endpoint tumor volumes were determined on dissected masses at the time of necropsy. c mRNA levels of mdm2 and mdmx normalized to gapdh in primary tumors were determined by real-time qRT-PCR. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = nonsignificant. The p value was calculated using two-tailed unpaired t tests. d Protein expression of MDM2, MDMX, and mtp53 from 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx engrafted primary tumors were determined by Western blot analysis. Three tumors per group were used, and actin is shown as a loading control

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Control

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 provoke in vitro MDA-MB-231 cell migration without altering cell proliferation. a Representative Western blot demonstrating the levels of MDM2, MDMX, and mtp53 in 231.mir30.vector, 231.sh mdm2 , and 231.sh mdmx cells (lanes 1, 2, and 3, respectively). Fifty micrograms of lysate was loaded per lane. Actin was used as a loading control. b The number of cells was determined by hemocytometer cell counting. Cells ( n = 50,000) were seeded in triplicate, and cell counting was performed at 2, 4, 5, and 6 days. Dots represent mean values, and error bars represent SD. Experiments were carried out with three biological replicates. c Wound closure was observed by phase-contrast microscopy and photographed at 0 and 12 h. One representative image from each group at 0 and 12 h is shown. d The wound area was measured by using NIS-Elements software (Nikon Instruments, Melville, NY, USA). The percentage of wound closure was quantified from four independent biological experiments. The p value was obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: In Vitro, Migration, Western Blot, Plasmid Preparation, Control, Cell Counting, Microscopy, Software, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX knockdown in primary tumors blocks the transcription of CXCR4 and PTGS2 . The 231.mir30.vector-, 231.sh mdm2 -, and 231.sh mdmx -derived primary tumors were lysed and used for total RNA extraction and complementary DNA synthesis. a Microarray analysis revealed selected tumor metastasis-related genes that were either up- or downregulated in 231.sh mdm2 and 231.sh mdmx compared with 231.mir30 vector. Fold changes were gated either > 2 or < 0.5. Two tumor samples per group were used for the analysis. b From the respective cells derived from all the primary tumors, the total CXCR4 and PTGS2 levels were determined by real-time qRT-PCR, and these were compared with those of the parental cells grown in culture. The bars represent mean values, and error bars represent SD. The p values were obtained by two-tailed unpaired t test

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, Plasmid Preparation, Derivative Assay, RNA Extraction, DNA Synthesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDM2 knockdown in ERα + T47D orthotopic transplant reduces tumor volume. a T47D cells with inducible sh mdm2 or mir30 shRNA-expressing control vector were treated with 4 μg/ml doxycycline (Dox) for 10 days to induce and maintain shRNA expression. Western blot shows the levels of MDM2, MDMX, E-cadherin, and mtp53 with Dox treatment (lanes 1 and 2) prior to mammary fat pat implantation. Actin was used as a loading control. b Animals were provided with 2 mg/ml Dox and 8 μg/ml E 2 in their drinking water during the entire experiment. Primary tumor growth was measured over a period of 60 days using calipers *** p < 0.001 calculated by two-tailed unpaired t test. c The experimental endpoint tumor volume was determined at the time of necropsy. d mdm2 mRNA expression in primary tumors was determined by real-time qRT-PCR. The p value was determined by two-tailed unpaired t test. e E-cadherin, MDM2, MDMX, and mtp53 protein levels from primary tumors were determined by Western blot analysis. Actin was used as the loading control. f Representative H&E staining images of T47D.vector and T47D.sh mdm2 under 200× and 1000× magnification. T represents Tumor; nm represents normal mammary fat pad; M represents muscle; arrowhead depicts tumor cells infiltrating into muscle layer

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Knockdown, shRNA, Expressing, Control, Plasmid Preparation, Western Blot, Two Tailed Test, Quantitative RT-PCR, Staining

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: Comparative levels of CXCR4 in ERα + T47D and TNBC MDA-MB-231 tumors. a CXCR4 RNA expression normalized to gapdh with MDM2 knockdown was determined using real-time qRT-PCR. 231.mir30.vector ( n = 6), T47D.mir30.vector ( n = 10), and T47D.sh mdm2 ( n = 9) tumor samples were analyzed. The RNA level of CXCR4 was set as 1 for the 231.mir30.vector group, and T47D samples were expressed relative to 231.mir30.vector values. b Protein expression of CXCR4 was compared from parental cell lines and two tumors from each group. Representative Western blot demonstrating protein levels of CXCR4 and actin in MDA-MB-231 and T47D groups shown using two gels (lanes 1–9 and 10–16, with tumor 2 for 231.sh mdmx used in lane 10 as a common reference)

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: RNA Expression, Knockdown, Quantitative RT-PCR, Plasmid Preparation, Expressing, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

doi: 10.1186/s13058-018-1094-8

Figure Lengend Snippet: MDMX and MDM2 in TNBC promote metastasis, and in ERα + breast cancer MDM2 promotes proliferation. In TNBC, MDMX promotes expression of CXCR4 and PTGS2 with associated release of CTCs but no increase in cell proliferation. In ERα + breast cancer, estrogen stimulates MDM2 expression with no influence on CXCR4 and causes an increase in cell proliferation without correlated metastasis

Article Snippet: Human breast cancer cell lines T47D ( mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 ( mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection ( www.atcc.org ; Manassas, VA, USA).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: Cavin4b/Murcb Is Required for Skeletal Muscle Development and Function in Zebrafish

doi: 10.1371/journal.pgen.1006099

Figure Lengend Snippet: Inset: PANTHER Database protein class statistical overrepresentation test on microarray mRNA expression profiling from murcb s983/s983 compared to murcb s983/+ 72 hpf larvae. 1309 genes with greater than 1.5 fold or less than 0.7 fold expression difference in Cavin4b/Murcb deficient larvae compared to their heterozygous siblings were recognized in the database with 25708 genes comprising the Danio rerio reference list. Protein classes shown had a p value <0.05. The log 2 ratio of murcb s983/s983 / murcb s983/+ normalized microarray values from genes in the voltage-gated ion channel PANTHER protein class show that 26 members of the voltage-gated ion channel class were misregulated where 10 would be expected. Error bars represent SEM.

Article Snippet: RNA expression from heterozygous and mutant larvae was analyzed by single-color microarray (8x60K Zebrafish Array XS, Oaklabs, Germany).

Techniques: Microarray, Expressing