3× flag fusion proteins  (Millipore)

Journal: Cell Death Discovery

Article Title: CircPPFIA2 drives prostate cancer progression and enzalutamide resistance by sponging miR-646 and miR-1200 to upregulate ETS1

doi: 10.1038/s41420-025-02904-z

Figure Lengend Snippet: A Venn diagram identifies miR-646, miR-1200, and miR-581 as top candidate miRNAs targeting circPPFIA2. B Luciferase reporter assays validate miR-646/miR-1200-mediated suppression of circPPFIA2 activity in HEK-293T cells. C Biotin-tagged circPPFIA2 or control probes were used in pull-down assay of miRNAs in C4-2 cell. D qRT-PCR analysis of circPPFIA2 enrichment in Flag-Ago2 immunoprecipitates, with IgG as the negative control. n = 3 biological replicates; n = 3 biological replicates; values are shown as % input normalized to IgG. E Schematic of circPPFIA2 reporters showing conserved miR-1200 (bp 188–194) and miR-646 (bp 323–329) binding sites; the mutant contains point mutations at these miRNA-binding sites. F , G Dual-luciferase assays showing that co-transfection of miR-1200/miR-646 mimics reduced WT reporter activity; this effect was abolished in the mutant reporter with miRNA-binding-site mutations. n = 3 biological replicates; values are firefly normalized to Renilla and to miR-NC. H RNA-FISH confirms cytoplasmic co-localization of circPPFIA2 with miR-646/miR-1200. Scale bar: 5 μm. I – N Rescue experiments demonstrate miR-646/miR-1200 inhibitors reverse circPPFIA2 silencing effects, whereas mimics counteract circPPFIA2-driven oncogenicity. Scale bar: 50 μm. Data are presented as mean ± SD ( n = 3 biological replicates). Two-tailed tests with Bonferroni correction were used; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the Ago2–circPPFIA2 assay, C4-2 cells stably expressing FLAG-Ago2 were generated by lentiviral transduction followed by puromycin selection; clarified lysates (~1–2 mg total protein per IP) were incubated with rabbit anti-FLAG (Proteintech, Cat. 20543-1-AP, 5 μg per IP) or normal rabbit IgG pre-bound to Protein G magnetic beads (~25 μL per IP) for 3 h at 4 °C with rotation.

Techniques: Luciferase, Activity Assay, Control, Pull Down Assay, Quantitative RT-PCR, Negative Control, Binding Assay, Mutagenesis, Cotransfection, Two Tailed Test

Journal: Advanced Science

Article Title: SOSTDC1 Nuclear Translocation Facilitates BTIC Maintenance and CHD1‐Mediated HR Repair to Promote Tumor Progression and Olaparib Resistance in TNBC

doi: 10.1002/advs.202306860

Figure Lengend Snippet: Nuclear SOSTDC1 interacts with CHD1 to promote HR repair, BTIC maintenance, and Olaparib resistance. A) Co‐IP assay detected the interaction between SOSTDC1 and CHD1. Vectors encoding HA‐tagged CHD1 or FLAG‐tagged SOSTDC1 were transfected into 293T cells, which were subjected to co‐IP experiments with magnetic beads coated with antibodies to FLAG or HA, followed by western blotting with the indicated antibodies. B) In vitro co‐IP assay detected the interaction between SOSTDC1 and CHD1. Purified HA‐tagged SOSTDC1 or FLAG‐tagged CHD1 recombinant proteins were subjected to co‐IP experiments with anti‐FLAG magnetic beads, followed by western blotting with the indicated antibodies. C) Co‐IP assay detected the interaction between SOSTDC1 and full length CHD1 (fl) or its three truncated mutants (a, b, c). D) Immunofluorescence analysis of the distribution of endogenous SOSTDC1 and CHD1 in SUM149 cells upon 1 µmol L −1 Olaparib treatment for 12 h. Scale bar, 5 µm. E) Co‐IP assay detected the interaction between nuclear SOSTDC1 and CHD1. Cytoplasmic and nuclear lysates from FLAG‐tagged SOSTDC1‐overexpressing SUM159 cells were prepared for co‐IP experiments with anti‐FLAG magnetic beads, followed by western blotting with the indicated antibodies. F) Time‐dependent analysis of the expression of CHD1 by western blotting after 1 µmol L −1 Olaparib treatment in SUM159 cells. G) Co‐IP assay detected the interaction between SOSTDC1 and CHD1 upon 1 µmol L −1 Olaparib treatment for 12 h. H) CHD1 protein expression in sorted BTICs (B) and the rest non‐BTICs (R) of SUM149 and SUM159. I) Immunohistochemistry analysis of CHD1 expression in para‐tumor tissues and tumor tissues from BC patients. Representative images of different molecular subtypes were shown and H‐Scores were calculated. Data were presented as mean ± SEM. Significance was calculated using unpaired Student's two‐sided t ‐tests. * p < 0.05; *** p < 0.001; ns, not significant. Scale bar, 100 µm. J) Analysis of the correlation between SOSTDC1 protein levels (related to Figure ) and CHD1 protein levels (related to Figure 4I) in BC patient tumor tissues. Statistical significance was determined by Pearson's correlation test. K) Immunohistochemistry analysis of CHD1 expression in SOSTDC1‐knockdown SUM149 xenografts. Representative images were shown and H‐Scores were calculated. Data were presented as mean ± SEM, following unpaired Student's two‐sided t ‐tests. ** p < 0.01. Scale bar, 100 µm. L) SOSTDC1‐overexpressing SUM159 cells with CHD1‐knockdown were treated with DMSO or Olaparib (0.1 µmol L −1 , 1 µmol L −1 ) for 12 h, following immunofluorescence analysis of γ‐H2AX foci. Percentages of γ‐H2AX foci‐positive cells (>10 foci) were quantified. Data were presented as mean ± SEM, following the one‐way ANOVA. * p < 0.05; ns, not significant. M) Analysis of HR efficiency in SOSTDC1‐overexpressing cells with CHD1‐knockdown using the DR‐GFP reporter assay. Data were presented as mean ± SEM. Significance was calculated using the one‐way ANOVA. * p < 0.05, ** p < 0.01. N) Mammosphere formation assays of SOSTDC1‐overexpressing SUM159 cells with CHD1‐knockdown. Data were presented as mean ± SEM, following the one‐way ANOVA. *** p < 0.001. Scale bar, 100 µm. O) The percentage of BTIC population in SOSTDC1‐overexpressing SUM159 cells with CHD1‐knockdown. Data were presented as mean ± SEM, following the one‐way ANOVA. *** p < 0.001. P) Cell viability determined by MTT assay in SOSTDC1‐overexpressing SUM159 cells with CHD1‐knockdown. n = 3 independent biological samples.

Article Snippet: Proteins were then eluted by competitive elution of 3× FLAG fusion proteins (0.4 mg ml −1 ; F4799, Sigma‐Aldrich) and boiled or directly denatured in 2× loading buffer.

Techniques: Co-Immunoprecipitation Assay, Transfection, Magnetic Beads, Western Blot, In Vitro, Purification, Recombinant, Immunofluorescence, Expressing, Immunohistochemistry, Knockdown, Reporter Assay, MTT Assay