Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: Western blotting, quantitative RT-PCR and IHC assay of the expression of SLC34A2 in BC cell lines and tissues. ( a and b ) Western blotting (upper panel) and quantitative RT-PCR (lower panel) assay of SLC34A2 expression in ( a ) four BC cell lines and three normal bladder tissues (N), and in ( b ) five pairs of matched BC (T) and adjacent non-neoplastic bladder tissue (ANT). The average expression level of SLC34A2 in N or ANT was used as a loading control in quantitative RT-PCR assay. ( c-f ) Representative image of SLC34A2 IHC staining in BC tissues: negative staining in normal bladder tissue ( c ), and weak ( d ), moderate ( e ) and strong ( f ) staining in BC tissues, respectively. Images are presented at × 200 magnification

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Control, Immunohistochemistry, Negative Staining, Staining

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: The selection of optimal cutoff value for SLC34A2 IHC score in two different cohorts of BC samples. ( a ) X-tile analysis was conducted on patient data from the training cohort, equally subdivided into training and validation subsets. X-tile plots of training sets are displayed in the left panels, with matched validation sets in the smaller inset. The plot showed the χ 2 log-rank values created when the cohort was divided into two populations. The cut point was demonstrated on a histogram of the entire cohort (middle panels) and a Kaplan–Meier plot (right panels). P- values were defined by using the cut point derived from a training subset to parse a separate validation subset. SLC34A2 expression was divided at the optimal cut point (for high expression, n =82; for low expression, n =74), where the plot achieves highest significance (with positive staining of SLC34A2; P <0.001). ( b ) The optimal cut point for SLC34A2 expression determined by X-tile plot of the testing cohort was applied to the validation cohort (for high expression, n =71; for low expression, n =59) and reached high statistical significance ( P <0.001). ( c ) ROC curve analysis for different clinicopathological features and SLC34A2 expression was performed to evaluate the survival status. Left panel: age (AUC=0.455; P =0.338), gender (AUC=0.539; P =0.406), grade (AUC=0.533; P =0.478), tumor size (AUC=0.586; P =0.064), T statue (AUC=0.565; P =0.166), N statue (AUC=0.540; P =0.390), tumor multiplicity (AUC=0.583; P =0.046) and SLC34A2 expression (AUC=0.658; P =0.001) implied statistical associations with survival in the training cohort; Right panel: age (AUC=0.446; P =0.289), gender (AUC=0.492; P =0.880), grade (AUC=0.592; P =0.069), tumor size (AUC=0.654; P =0.002), T statue (AUC=0.623; P =0.015), N statue (AUC=0.569; P =0.173), tumor multiplicity (AUC=0.585; P =0.050) and SLC34A2 expression (AUC=0.692; P <0.001) were used to test the survival status in validation cohort

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: Selection, Biomarker Discovery, Derivative Assay, Expressing, Staining

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: Association of SLC34A2 expression with patient's clinicopathological features in BC

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: Expressing, Biomarker Discovery

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: Univariate and multivariate analysis of SLC34A2 expression and various clinicopathological parameters in training and validation cohort patients with BC

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: Expressing, Biomarker Discovery

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: Effects of SLC34A2 on cell proliferation in vitro . ( a ) The protein expression of SLC34A2 analyzed by western blotting in shSLC34A2 transduced EJ and T24 cells compared with scramble controls, and SLC34A2 stably overexpressed 5637 cells compared with negative control cells. ( b and c ) EJ and T24 cells with reduced SLC34A2 showed significant growth inhibition compared with scramble controls, and 5637 cells with upregulated SLC34A2 proved growth promotion compared with controls, as determined by ( b ) MTT assays and ( c ) colony formation assays. ( d ) Downregulation of SLC34A2 greatly suppressed the anchorage-independent growth of both EJ and T24 cells, but upregulation of SLC34A2 significantly accelerated the malignancy transforming growth of 5637 cells. Colonies in ( c ) and ( d ) larger than 0.1mm diameter were quantified after 14 days of culture. ( e ) Left panel: expression of SLC34A2 is positively associated with Ki-67 in clinical BC specimens. Two representative cases are shown. Right panel: percentage of specimens showing low or high SLC34A2 expression in relation to the expression levels of Ki-67. * P <0.05

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: In Vitro, Expressing, Western Blot, Stable Transfection, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: Effects of SLC34A2 on tumor growth in vivo . ( a ) The xenograft tumor volumes of nude mice injected with EJ/scramble cells or EJ/shSLC34A2 cells ( n =6), and that of nude mice injected with 5637/control cells or 5637/SLC34A2 cells ( n =5). ( b ) The average tumor weight in nude mice injected with EJ/shSLC34A2 cells were significantly decreased compared with the EJ/scramble group ( n =6), whereas 5637 cells with overexpressed SLC34A2 largely increased the tumor burden ( n =5). * P <0.05

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: In Vivo, Injection, Control

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: Depletion of SLC34A2 suppressed the expression and transcription capacity of c-Myc. ( a ) The luciferase reporter activities of c-Myc were remarkably decreased in SLC34A2-silenced EJ and T24 cells. ( b and c ) In SLC34A2 stably reduced EJ and T24 cells, the protein expression of c-Myc was decreased as determined by ( b ) western blotting analysis and ( c ) immunofluorescence staining. ( d ) Left panel: expression of SLC34A2 is positively associated with c-Myc in clinical BC specimens. Two representative cases are shown. Right panel: Pearson's correlation coefficients analysis showed that SLC34A2 positively correlated with c-Myc expression in BC samples. ( e ) In both EJ and T24 cells, endogenous c-Myc were greatly downregulated in the presence of shSLC34A2–1, the suppression was reversed by inducing exogenous c-Myc. ( f-g ) The inhibition of cell viability and proliferation ability by shSLC34A2 was significantly compromised by treatment with exogenous c-Myc, as determined by ( f ) MTT assays and ( g ) colony formation assays. * P <0.05

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: Expressing, Luciferase, Stable Transfection, Western Blot, Immunofluorescence, Staining, Inhibition

Journal: Cell Death & Disease

Article Title: Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity

doi: 10.1038/cddis.2017.13

Figure Lengend Snippet: MiR-214 is a negative regulator of SLC34A2. ( a ) Illustration of screening miRNAs regulating SLC34A2 : Method #1 used the prediction algorithm of MICRORNA.ORG identified 23 miRNAs, which hold potential as SLC34A2 suppressors. Method #2 used miRNA profiling and identified 17 miRNAs, which were downregulated significantly in BC. Then, we overlapped the results of two methods and singled out miR-214 as a potential regulator. ( b ) Quantitative RT-PCR assay of miR-214 expression in four BC cell lines and three normal bladder tissues (N) (left panel), and in five pairs of matched BC (T) and adjacent non-neoplastic bladder tissue (ANT) (right panel). The average expression level of miR-214 in N or ANT was used as a loading control in quantitative RT-PCR assay. ( c ) Predicted miR-214 target sequence in 3′-UTR of SLC34A2 (SLC34A2 3′-UTR) and mutant miRNA containing three altered nucleotides in the seed sequence of miR-214 (miR-214-mut). ( d ) Luciferase assay of pGL3-SLC34A2 3′-UTR reporters co-transfected with increasing amounts (10, 20 and 50 nM) of miR-214 mimic or miR-214-mut in EJ and T24 cell lines. ( e ) Western blotting analysis demonstrates that miR-214 transfection markedly decreased SLC34A2 protein levels of EJ and T24 cells, whereas miR-214-mut exerted no inhibition effect. * P <0.05

Article Snippet: The two shRNA used to repress SLC34A2 expression and the coding sequences of SLC34A2 expression vector are purchased from GeneCopoeia (Guangzhou, China).

Techniques: Quantitative RT-PCR, Expressing, Control, Sequencing, Mutagenesis, Luciferase, Transfection, Western Blot, Inhibition

Journal: Human Genomics

Article Title: Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis

doi: 10.1186/s40246-022-00387-y

Figure Lengend Snippet: Overview of the SLC34A2 variants investigated

Article Snippet: The variants were introduced into a commercially available c-Myc-DDK-tagged SLC34A2 vector, in which the c-Myc epitope was fused to the C-terminus of the cotransporter in a pCMV6 backbone (OriGene Technologies, Rockville, MD, USA) (NCBI GenBank accession no. NM_006424 and AF067196), using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Correlation between renal Rn Slc34a2 expression (by quantitative RT-PCR analysis) and fasting urinary Si excretion. Urinary Si excretion in the rats (■, Si deplete; ●, Si replete) and laboratory chow reference group (▲, Si-high reference) showed an inverse relationship with Slc34a2 expression in the kidneys; r = 0.47.

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Expressing, Quantitative RT-PCR

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Immunohistochemistry analysis of Slc34a2 in freshly harvested rat kidney cortex. Sections of freshly harvested kidneys from a healthy wild-type rat were analyzed by immunohistochemistry with anti-Slc34a2 ( green ) antibody (this figure) or the appropriate isotype control (data not shown). Cell nuclei were counterstained ( blue ) with Hoescht 33342 and cell cytoskeleton (f-actin, red ) with phalloidin CF633. Antibody-stained sections and isotype controls were collected under identical settings, as stated in materials and methods . A threshold removing all Slc34a2 attributable signal was defined on the isotype controls and uniformly applied to all images (i.e., antibody-stained images). Staining for Slc34a2 within the tubular epithelial cells was distinctly punctate so, as well as the signal above the isotype control being presented in an as-collected “intensity” format (i.e., the more secondary antibody that is bound, the brighter the signal) ( A and B ), it is also displayed as a binary format (i.e., all signal that is brighter than isotype threshold is given the maximum intensity value) as this aids visualization ( C and D ). All images are of the kidney cortex and scale bars are 50 µm. B : as-collected “intensity” format without actin staining. D : a high-power image (×63 magnification) of the area within the quadrant in image ( C ).

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Immunohistochemistry, Control, Staining

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Identifying Rn Slc34a2 as a candidate for Si transport. A : relative expression of solute-like carriers in the kidney of Rattus norvegicus from Si deplete ( n = 4) compared with Si replete ( n = 4) animals. Data were analyzed by gene array. Red indicates upregulation and blue indicates downregulation of expression; Si replete vs. Si deplete group. Multiple probe sets per gene can be present as was the case for Slc13a1. B : quantitative PCR analysis of Slc34a2 and of family members ( inset ) in the kidneys of Si-high reference, Si replete and Si deplete rats. Overall, the relative expression of Rn Slc34a2 was inversely related to dietary Si exposure ( P < 0.05), but there was no relationship with Slc34a1 or Slc34a3 ( P = 0.5 and 0.4, respectively). Gene expression values are relative to the Si-high reference group. C : sequence alignment of the Rattus norvegicus Slc34 gene family. Slc34a2 is characterized by a ~30-residue stretch (highlighted in yellow) that is not present in Scl34a1 and Slc34a3. Asterisks (*) below sequence indicate identical amino acids, colons (:) indicate functionally similar amino acids, and dashes (–) indicate gaps in the alignment.

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Sequencing, Residue

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Fasting urinary phosphorus excretion. Urinary P excretion was measured in the laboratory chow reference group (Si-high reference; n = 6), Si replete ( n = 5), and Si deplete ( n = 8) rats by ICP-OES and corrected for creatinine concentration ( A ). The higher P excretion in the laboratory chow reference group is due to the higher P content of the diet (see B ). However, the difference in urinary P excretion between the Si replete and Si deplete rats cannot be explained by a difference in dietary P content, but rather due to the upregulation of Slc34a2 in the latter group mediated by Si deficiency in the diet and drinking water ( B ).

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Transport activity in Rn Slc34a2-expressing oocytes. A–C : influx transport activity of Rattus norvegicus Slc34a2 for arsenate, H A s O 4 2 − ( P = 0.0001) ( A ), phosphate, H P O 4 2 − ( P = 0.0008) ( B ), and silicic acid, H 4 SiO 4 ( P = 0.66) ( C ) . Rice transporter Lsi1 was used as a positive control for H 4 SiO 4 influx ( P < 0.0001). D : the concentrations of sodium and phosphate in the medium did not influence H 4 SiO 4 influx by Rn Slc34a2-expressing oocytes, nor that by Os Lsi1-expressing oocytes ( P < 0.0001 in both instances). Water-injected oocytes were used as a negative control. E : in H 4 SiO 4 efflux studies, rice transporter Lsi2 was used as a positive control. Data were corrected against water-injected control oocytes. F : changes in sodium and phosphate concentration did not affect H 4 SiO 4 efflux by Slc34a2 expressing oocytes. Data are shown as means ± SE ( n = 15).

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Activity Assay, Expressing, Positive Control, Injection, Negative Control, Control, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Pairwise alignment of the transmembrane domains of Si efflux transporters. Pairwise alignment of the transmembrane domains predicted in Rn Slc34a2 rat protein (red) with the four Si efflux transporters in plants (green). Transmembrane domains were predicted by OCTOPUS , and subsequent alignment was performed by AlignMe tool . A : Os Lsi2 (rice); B : Zm Lsi2 (maize); C : Hv Lsi2 (barley); D : Cm Lsi2–1 (pumpkin).

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Phylogeny of Slc34a gene family member in vertebrates. A : the tree was produced using PhyML maximum likelihood analysis with the JTT+G+I model from an alignment of 880 positions. Numbers at nodes are a percentage of 100 bootstrap replicates, with nodes having <70% bootstrap support being collapsed. B : the tree was produced using Phylobayes Bayesian MCMC analysis under the CAT +G+I model (15 parallel chains with sampling every 100 cycles, burn-in one-fifth the total size of the chain) from an alignment of 880 positions. Numbers at nodes indicate posterior probabilities, with nodes having <0.95 support being collapsed. The scale bar indicates the average number of amino acid substitutions per site. The Slc34a1 clade is in green, the Slc34a2 clade is in blue, and the Slc34a3 clade is in red. The trees are rooted using the single Slc34a homolog identified from the lamprey genome. The Slc34a gene phylogeny largely agrees with the species phylogeny for vertebrates , with incongruent branches (e.g., the basal branches of the a2 clade) only having low statistical support. The maximum likelihood phylogenetic analyses resolve that the Slc34a clade evolved from a single ancestor in jawless vertebrates, and likely involved two main duplication events, initially producing the a3 and a1+2 clades, with a further divergence of the a1 and a2 clades. A teleost-specific duplication event resulted in the evolution of Slc34a2a and Slc34a2b, as found in stickleback and zebrafish. The Bayesian analysis had poor phylogenetic resolution at the base of the a2 clade, but still resolves the a1 and a3 groups as distinct monophyletic clades, and is not incongruous with the maximum likelihood analysis results. For full details of the species and sequences used see Supplemental Table S1.

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Produced, Sampling

Journal: American Journal of Physiology - Cell Physiology

Article Title: Identification of a mammalian silicon transporter

doi: 10.1152/ajpcell.00219.2015

Figure Lengend Snippet: Alignment of vertebrate Slc34a protein sequences showing characteristic motif conserved across members of the Slc34a2 group. The alignment shows the region around the portion identified as unique to rat Slc34a2 in comparison to rat Slc34a1 or Slc34a3 (see Fig. 1 C ). Highlighted in yellow are the homologous regions in other vertebrate Slc34a2 proteins, and in the Slc34a-type lamprey sequence. The characteristic Slc34a2 motif identified within this region contains at least three positive residues uninterrupted by any negatively charged residues, with the positive residues regularly spaced apart by at least four small residues (primarily cysteines). A Slc34a sequence containing this motif was found in all vertebrate species investigated. The only members of the Slc34a2 clade (see ) where this motif was incomplete was are in the zebrafish and stickleback SLC34a2b (highlighted in blue). Positively charged residues are shown in bold and small amino acids are in italics. Sequence names correspond to the species and gene identifiers given in Supplemental Table S1 and to the phylogeny shown in . The incomplete spiny shark and skate NaPi-IIb sequences are omitted due to this region being missing from the EMBL/GenBank data. The alignment was generated using MUSCLE ( http://www.ebi.ac.uk/Tools/msa/muscle/ ).

Article Snippet: Samples were then incubated with primary antibody against Slc34a2 (Genetex) or an appropriately matched isotype control.

Techniques: Comparison, Sequencing, Generated

Journal: Diagnostics

Article Title: Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants

doi: 10.3390/diagnostics12030729

Figure Lengend Snippet: Control charts for CNA and fusion/skipping variant types. ( a ) CCDC6-RET fusion, ( b ) EML4-ALK fusion, ( c ) CD74-ROS1 fusion, ( d ) ERBB2/HER2 amplification, and ( e ) MYC amplification detected within the positive control over seven days of testing.

Article Snippet: HCC78 harboring SLC34A2-ROS1 (Cat #: CSC-C0569; Creative Bioarray, NY, USA), CRL-5935 harboring EML4-ALK (Cat #: NCI-H2228; ATCC, Manassas, VA, USA), KM12 (acquired from MD Anderson Cancer Center, Houston, TX, USA) harboring TPM3-NTRK1, and HTB-178 (Cat #: NCI-H596, ATCC) harboring MET exon 14 skipping were used for fusions and exon skipping.

Techniques: Variant Assay, Amplification, Positive Control

Journal: Diagnostics

Article Title: Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants

doi: 10.3390/diagnostics12030729

Figure Lengend Snippet: Analytic sensitivity for the fusion/skipping variant type.

Article Snippet: HCC78 harboring SLC34A2-ROS1 (Cat #: CSC-C0569; Creative Bioarray, NY, USA), CRL-5935 harboring EML4-ALK (Cat #: NCI-H2228; ATCC, Manassas, VA, USA), KM12 (acquired from MD Anderson Cancer Center, Houston, TX, USA) harboring TPM3-NTRK1, and HTB-178 (Cat #: NCI-H596, ATCC) harboring MET exon 14 skipping were used for fusions and exon skipping.

Techniques: Variant Assay