Journal: Cancer Research Communications

Article Title: BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma

doi: 10.1158/2767-9764.CRC-21-0047

Figure Lengend Snippet: GSEA analysis and validation of a primary cell line from a Ewing tumor with a BARD1 pathogenic variant. A, Schematic overview of tumor samples associated with analyses in B – F . B, Gene-set enrichment analysis (GSEA) of RNA-seq data comparing the lung relapse of the Ewing tumor with a germline BARD1 pathogenic variant to the original primary/pretreatment biopsy. Genesets significantly impacted ( P < 0.05) are included. C, Phase contrast image (400×) of the PSaRC318 Ewing tumor cell line. D, Flow cytometry showing presence of surface CD99 expression in the PSaRC318 cell line. E, Schematic detailing the difference between Type 1 and Type 3 EWS-FLI fusions (top) and Western blot analysis with anti-FLI1 antibody of Ewing sarcoma cell lines with type 3 (PSaRC318) versus type 1 (A673, CHLA9, CHLA10, and TC71) EWS-FL1 fusions. F, Western blot demonstrating BARD1 protein expression in the same Ewing sarcoma cell lines as in E . PSaRC318 cells demonstrate significantly ( P < 0.05) less BARD1 expression as compared with other Ewing cell lines. Densitometry values below the blot indicate relative expression values. Experiments in D – F were completed minimally in biological triplicate.

Article Snippet: Antibodies were purchased from the following sources: anti-CD99 FITC conjugated (BD Biosciences, catalog no: 555688, concentration 1:20), anti-FLI1 (Abcam, catalog no: 133485, concentration 1:2,000), anti-BARD1 (Bethyl Laboratories, catalog no: A300–263A, 1:2,000), anti-BARD1 (Abcam, catalog no: ab50984, concentration 1:100), anti-GBP1 (Abcam, catalog no: 131255, concentration 1:300 for IHC), anti-GBP1 (Santa Cruz Biotechnology, catalog no: sc-53857, concentration 1:200 for Western blot analysis), anti-phospho (Ser 139)-γH2A.X (Millipore Sigma, catalog no: 05–636, concentration 1:2,500 for immunofluorescence), anti-phospho (Ser 139)-γH2A.X (Invitrogen, catalog no: MA1–2022, concentration 1:1,000 for Western blot analysis), goat anti-mouse IgG AF-488 (Thermo Fisher Scientific, catalog no: A-11001, concentration 1:2,000), tubulin (Cell Signaling Technology, catalog no: 2144S, concentration 1:5,000), vinculin (Cell Signaling Technology, clone E1E9V, catalog no: 13901S, concentration 1:5,000), and anti-rabbit IgG-horseradish peroxidase (HRP) (Promega, catalog no: W401B).

Techniques: Variant Assay, RNA Sequencing Assay, Flow Cytometry, Expressing, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Prolactin stimulates sodium and chloride ion channels in A6 renal epithelial cells

doi: 10.1152/ajprenal.00270.2014

Figure Lengend Snippet: PRL activates a 2-pS chloride channel in the apical membrane of A6 epithelia. A: the transepithelial current was calculated from transepithelial voltage and resistance measurements in polarized A6 renal epithelial cells grown on permeable supports before and 30 min after application of vehicle or Inhibitor-172, a putative CFTR inhibitor (10 μM). At this point, we added vehicle or 1 μg/ml PRL to the basolateral media and monitored the transepithelial current for 60 min and then 15 min after 2 μM amiloride application. Inhibitor-172 dramatically reduced the amiloride-insensitive portion of the PRL-induced transepithelial current in A6 cells. Arrows point to approximate time of inhibitor application; n = 3/group. Values are means ± SE by 2-way repeated measures ANOVA to detect significant differences with a post hoc Student-Newman-Keuls test. *P < 0.05 vs. vehicle at a given time point. **P < 0.05 vs. PRL at a given time point. B: to confirm the identity of the chloride channel induced by PRL, we performed cell-attached patch clamp in A6 cells. Both ENaC and the chloride channels are present in the records. The chloride channels are marked with arrows and distinguishable from ENaC by their small currents and relatively short mean open times. Dashed lines indicate the channel closed state. C: current-voltage relationship for the chloride channel detected in patches after application of PRL to the basolateral media. Vp = pipette holding potential. The line through the data is the best nonlinear least-squares fit to the Goldman-Hodgkin-Katz equation. D: Po for chloride channels in B depends upon voltage, as shown in this plot (normalized to maximum Po). E: RT-PCR (32 cycles) was run on cDNA from A6 (2F3) cells and frog (Xenopus laevis) kidney with primers specific to either ClCn4 or ClCn5. Electrophoresis of the resulting PCR products (expected at ∼500-bp size) demonstrated high levels of transcripts for ClCn4. However, we observed no amplification of ClCn5 transcripts from either the cells or kidney samples. NTC, no template control containing all primers for ClCn4 and ClCn5, showed no amplification of a PCR product. F: a typical Western blot of ClC4 in 2F3 cells. After 8 days in culture, in the presence of standard 2F3 with aldosterone, cells were fed with aldosterone-free media overnight. On day 9, 3 groups of cells were treated with standard media (C), sterile water (W), or 1 μg/ml PRL and incubated for 24 h before all 3 were lysed and blotted.

Article Snippet: We used an antibody generated to a KLH-conjugated synthetic peptide between 663 and 689 amino acids from the C-terminal region of human CLC4 (Pierce Antibody PA5-13348).

Techniques: Membrane, Patch Clamp, Transferring, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Amplification, Control, Western Blot, Sterility, Incubation