Journal: Nature

Article Title: Reduced cyclin D3 expression in erythroid cells protects against malaria

doi: 10.1038/s41586-026-10110-9

Figure Lengend Snippet: a , Top, vector content and expression of luciferase reporter genes driven by the extreme allele combinations: on the one hand the WT rs9349205-G|rs112233623-C (G|C) alleles on the other hand the derived rs9349205-A| rs112233623-T (A|T) alleles of the CCND3 enhancer in the erythroid cell line HUDEP-2, with empty vector as a control construct. Below, histograms show averages of the relative luminescence activity of the combinations of the two extreme alleles at rs9349205 and rs112233623 described above in absence (-) or presence of GATA1 and FOG-expressing plasmids; SMAD3 and p300 expressing plasmids; SMAD3 expressing plasmid alone; or empty expression vector. All values are plotted relative to the WT (G|C) construct. Note that the activity of the empty pGL4 vector is barely detectable. The mean ± s.e.m is shown (n = 7 (vectors); n = 3 (vectors + SMAD3), n = 3 (vectors+SMAD3 + p300), n = 4 (vectors + GATA1 + FOG) biologically independent experiments). A two-sided Student’s t -test was used, with level of significance indicated by asterisks (*P < .05;**P < .01; ****P < .0001, ns, not significant). The statistical results for all comparisons are provided in Supplementary Table . b , In silico prediction of binding to rs112233623 allele variants, showing derived allele T hindering binding of SMAD3 while favouring GATA1 binding. c , d , Representative electrophoretic mobility shift assays (EMSA) showing binding of SMAD3 and GATA1 proteins with labelled oligonucleotide probes (*) containing the WT (C) or derived (T) allele of rs112233623 (n = 3 biologically independent experiments). Competitor unlabelled oligonucleotides were used at the indicated fold excess to demonstrate specificity of binding. c , SMAD3 binds to the WT rs112233623-C allele (lanes 2 and 8); is weakly supershifted by anti-SMAD3 antibody (lanes 3 and 9); and is competed away by an excess of unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 10-12, at 25X, 50X and 100X respectively), but not by an excess of oligonucleotide containing the derived rs112233623-T allele (lanes 13-15, at 25X, 50X and 100X respectively). d , GATA1 binds to the rs112233623-T derived allele (lanes 2, 8 and 9), is supershifted by anti-GATA1 antibody (lane 3); and is competed away by an excess of unlabelled DNA oligonucleotides containing the derived rs112233623-T allele (lanes 10-12, at 25X, 50X and 100X respectively) compared to unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 13-15, at 25X, 50X and 100X respectively). GATA1 does not appear to bind to the WT rs112233623-C allele (lane 5). The samples derive from the same experiment and, the gels were processed in parallel. For gel source data, see Supplementary Fig. . e , f , ChIP–qPCR for SMAD3 or GATA1 binding to the CCND3 enhancer region surrounding rs112233623 ( CCND3 ) and to a SimpleChIP Human α Satellite as negative control in erythroblasts derived from individuals homozygous for the rs112233623-T decrease of expression (DoE) allele versus the WT rs112233623-C allele (WT). e , ChIP was performed using an antibody against SMAD3, results are represented as percentage of input and nonspecific IgG used as negative control. The mean ± s.e.m is shown (n = 3 biologically independent experiments). A two-sided two-Sample t-test was used; significant differences are indicated (*P < .05; ns, not significant). The statistical results for the comparisons are provided in Supplementary Table . f , ChIP assays were conducted with an antibody against GATA1, results are represented as percentage of input and nonspecific IgG used as negative control (n = 2 biologically independent experiments).

Article Snippet: ChIP DNA was purified and subsequently quantified by qPCR using primers designed to amplify a region surrounding rs112233623 in the CCND3 enhancer and SimpleChIP Human α Satellite Repeat Primers (4486, Cell Signaling Technology).

Techniques: Plasmid Preparation, Expressing, Luciferase, Derivative Assay, Control, Construct, Activity Assay, In Silico, Binding Assay, Electrophoretic Mobility Shift Assay, ChIP-qPCR, Negative Control

Journal: PLoS ONE

Article Title: Upregulation of Nuclear Factor-Related Kappa B Suggests a Disorder of Transcriptional Regulation in Minimal Change Nephrotic Syndrome

doi: 10.1371/journal.pone.0030523

Figure Lengend Snippet: A, AP1 DNA-binding activity of 25 microg of nuclear proteins. Nuclear extracts from NFRKB-transfected HEK cells were incubated with the wild-type AP1 oligonucleotide (Wt AP1) or with mutant-type AP1(Mut AP1) oligonucleotide. B, Expression of c-fos and c-jun. Immunoblots were performed with 50 microg of proteins. C, HEK cells were transiently co-transfected with the NFRKB or empty vector (Ev), and AP1-Luc reporter plasmid. The luciferase activity was measured using the “Dual Luciferase reporter Assay”. The data are presented as relative luciferase activity (firefly luciferase/the renilla luciferase). Statistical analyses were carried out on data from five independent experiments using the one-way Anova (** p <0.015; Kruskal-Wallis test).

Article Snippet: The double-stranded oligonucleotide probes (100 ng), with the consensus and mutant NF-kB (sc-2505, sc-2511), GAS/ISRE (sc-2537, sc-2538), NFATc (sc-2577, sc-2578) and AP1 (sc-2501, sc2514) sequences, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activity Assay, Transfection, Incubation, Mutagenesis, Expressing, Western Blot, Plasmid Preparation, Luciferase, Reporter Assay

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 3. RASSF10 modulated cell cycle. (a) Cell-cycle distribution was analyzed by FACS flow cytometry in QGY7703 cells and HepG2 cells stably transfected with pcDNA3.1-RASSF10 or pcDNA3.1 vector. Restoration of RASSF10 induced the accumulation of HCC cells in G1 cell cycle phase. The asterisk indicates statistical significance (*Po0.05). (b) Western blot shows the expression of major mediators in cell cycle process including p27, CyclinD1, CDK2 and CDK4.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: Cytometry, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 4. RASSF10 retarded tumor growth in vivo. (a) Subcutaneous tumor growth curve of RASSF10-expressing QGY7703 and HepG2 cells in nude mice was compared with vector (pcDNA3.1) transfected cells. The RASSF10 group showed a retarded tumor growth compared with the vector group (HepG2, P = 0.012; OGY7703, Po0.01). The data are means ± s.d. (n = 8/group). (b) A representative picture of tumor growth in nude mice subcutaneously inoculated with RASSF10 or vector (n = 8/group). (c) Histogram represents mean of the tumor weight from the RASSF10 and vector groups. The asterisk indicates statistical significance (*Po0.05, **Po0.01). (d) Cell cycle mediators including p27, Cycling D1, CDK2 and CDK4 were evaluated in the xenograft tumors by RT-PCR.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

doi: 10.1152/japplphysiol.00630.2016

Figure Lengend Snippet: Fig. 3. Basigin antibody validation. Using Western blotting, two duplicate gels (1 and 2) were loaded with four lanes of a single sample of skeletal muscle homogenate (M), six differ- ent concentrations of an internal standard (IS1– IS6: 8–38 g protein), and one lane of a Hep G2 cell lysate positive control (ve). Follow- ing transfer, membrane 1A was immunoblotted with a mouse monoclonal anti-basigin antibody (sc-21746), and membrane 2A was immuno- blotted with a different mouse monoclonal anti- basigin antibody (sc-46700). After imaging, the peroxidase of the horseradish peroxidase- conjugated secondary antibodies was inacti- vated by 15 min of H2O2 incubation. Both membranes were reprobed (1B and 2B) with a goat polyclonal anti-basigin antibody (sc- 9757). See text for additional details on immu- noblotting.

Article Snippet: Primary Rabbit polyclonal anti-MCT1 Merck Millipore AB3540P/2136555 1:1,000 1:15,000 Rabbit polyclonal anti-MCT4 Merck Millipore AB3316P/2397059 1:1,000 1:20,000 Mouse monoclonal anti-basigin Santa Cruz sc-21746/K1913 1:200 1:7,500 Mouse monoclonal anti-NHE1 Merck Millipore MAB3140/2283852 1:500 1:7,500 Rabbit polyclonal anti-NBCe1 Cell Signaling 11867/0001 1:500 1:5,000 Rabbit polyclonal anti-CAII Santa Cruz sc-25596/F0611 1:1,250 1:30,000 Mouse monoclonal anti-CAIII Abnova H00000761-M02/12243-S1 1:2,500 1:40,000 Mouse polyclonal anti-CAIV Abnova H00000762-B02P/08325 WULz 1:500 1:10,000 Mouse polyclonal anti-CAXIV Abnova H00023632-B01P/08358 WULz 1:750 1:10,000 Secondary Goat anti-mouse IgG Perkin Elmer NEF822001EA Goat anti-rabbit IgG Perkin Elmer NEF812001EA J Appl Physiol • doi:10.1152/japplphysiol.00630.2016 • www.jappl.org Overall basigin abundance changed in response to training (time main effect: F3,37.2 4.47, P 0.009) (Fig. 4C).

Techniques: Biomarker Discovery, Western Blot, Positive Control, Membrane, Imaging, Incubation