murine shrna  (Vector Biolabs)

Journal: Cancer Research Communications

Article Title: OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

doi: 10.1158/2767-9764.CRC-25-0696

Figure Lengend Snippet: Silencing of OPG in 67NR-primed CD19 + B cells abrogates their regulatory activity and restores 4T1 osteolytic and metastatic phenotypes in vivo . A, Experimental scheme: CD19 + B cells were isolated from the iliac BM of BALB/c mice bearing 67NR tumors on day 11 after implantation. Cells were subjected to gene silencing using lentiviral vectors carrying shRNA targeting Tnfrsf11b (OPG; B 67NR sh OPG) or a nontargeting scrambled control (B 67NR sh scr). Naive mice were used as experimental controls (Nv). B, OPG levels in culture supernatants from CD19 + B cells were assessed by ELISA. C, Experimental setup for in vivo analysis: OPG-silenced (sh OPG) or control-transduced (sh scr) CD19 + B cells were adoptively transferred together with CD3 + T cells from 4T1 tumor-bearing mice into immunocompetent BALB/c mice implanted with 1 × 10 5 4T1 tumor cells. D, Primary tumor growth was monitored over time. E, Representative images of tumor-bearing mice after adoptive B cell transfer. Photographs were taken at the experimental endpoint to illustrate macroscopic differences in tumor burden among groups. F, Quantification of RANKL levels by ELISA in supernatants of in vitro restimulated LNs and BM cells. G, Metastatic burden in draining LNs and BM was assessed by clonogenic assays. Data are presented as mean ± SD from two independent experiments ( n = 5 mice/group). Statistical comparisons between more than two groups were performed using one-way ANOVA followed by Tukey post hoc test for pairwise comparisons when data were normally distributed. Statistical significance was considered when P < 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To silence OPG expression in BM–derived CD19 + B cells from 67NR tumor–bearing BALB/c mice (day 11), cells were transfected with a specific murine short hairpin (sh) RNA (shRNA) plasmid targeting OPG [sc-40153-SH, Santa Cruz Biotechnology] or with a nontargeting control shRNA plasmid (sc-108060).

Techniques: Activity Assay, In Vivo, Isolation, shRNA, Control, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 promotes TNBC immune evasion. a Schematic diagram illustrating tumor/immune coculture-based CRISPRa screening used to identify genes that promote immune evasion. Py8119-OVA-dCas9-P65/HSF1 cells were used for screening. b The enrichment status of genes was plotted in comparison with that of the non-coculture group. c Py8119 cells labeled with ovalbumin and luciferase (Py8119-OVA-Luc) were used to generate stable endogenous FAM114A1-knockdown cell lines. Cells with stable FAM114A1-knockdown (KD#1 and KD#2) and the corresponding control (shCoo2) were collected for western blotting to test the efficacy of FAM114A1-knockdown. d , e Py8119-OVA-Luc cells with and without FAM114A1-knockdown were injected into the mammary fat pads of female OT-I mice. Tumor growth was monitored and measured weekly ( d ). The tumors were dissected, and tumor weight was measured at the endpoint ( e ). Representative tumors are shown ( e , left panel). n = 12 mice per group. Bar, 1 cm. f Tumors from the above groups were collected, formalin fixed, and paraffin embedded (FFPE). FFPE tumors were subjected to single-cell RNA sequencing. UMAP plots of the cell populations from the indicated groups. The T cells in each group are highlighted with red circles. g Average cell type proportions in the control (shCoo2) and FAM114A1-knockdown groups (FAM114A1-KD). The red start indicates the T-cell population. h Heatmap showing activated and nonactivated marker genes expressed in CD8 + T cells from the shCoo2 and FAM114A1-KD groups. i Fresh tumors from d were collected for flow cytometry analysis. The percentages of the indicated populations are shown. shCoo2, control group; KD#1 and KD#2, tumors with endogenous FAM114A1-knockdown. n = 6 tumors per group. j FFPE samples from TNBC patients were subjected to immunohistochemistry (IHC) staining with anti-FAM114A1 or anti-CD8 antibodies. Representative images are shown. The expression levels of FAM114A1 were quantified with ImageJ, and positive ratios of CD8 + T cells were determined. High- and low-FAM114A1 expression was determined by the median expression. Samples with ≥5% CD8 + T-cell infiltration were defined as CD8 + T-cell high, and those with <5% infiltration were defined as CD8 + T-cell low . n = 109 TNBC patients. Bar, 500 µm. The data represent the means ± SEMs. P-values were determined by one-way ANOVA ( d , e , i ) or the χ 2 test ( j )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Comparison, Labeling, Luciferase, Knockdown, Control, Western Blot, Injection, RNA Sequencing, Marker, Flow Cytometry, Immunohistochemistry, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 activates the PI3K/AKT pathway and suppresses antigen presentation. a Expression profile of cancer cells extracted from scFFPE-Seq of shCoo2- and FAM114A1-KD tumors. Gene set enrichment analysis (GSEA) was performed, and PI3K/AKT pathway enrichment in the shCoo2 group is shown. b scRNA-seq data of cancer cells extracted from TNBC patients. Cells without FAM114A1 expression were excluded, and the remaining cells were stratified into high- and low-FAM114A1 groups on the basis of median expression. PI3K/AKT activation scores (left panel) and antigen presentation signature scores (right panel) were calculated. c Py8119-OVA-Luc cell lines with (KD#1 and KD#2) and without (shCoo2) FAM114A1-knockdown were cocultured with OT-I splenocytes for 30 min. The tumor cells were isolated by sorting and subsequently collected for Western blotting. The expression levels of phosphorylated AKT (Ser473), total AKT, and FAM114A1 were tested. β-actin served as an internal control. d TNBC patient samples were analyzed by immunohistochemistry with anti-FAM114A1 and anti-phosphorylated AKT (Ser473) antibodies. Representative images are shown (left panel). The expression levels of FAM114A1 and phosphorylated AKT were quantified with ImageJ and analyzed with Spearman’s rank correlation (right panel). n = 109 TNBC patients. Bar, 500 µm. e Py8119 tumor cell lysates were collected and coimmunoprecipitated (co-IP) with anti-FAM114A1 (upper panel), anti-p85α (lower panel), or IgG controls. The interaction between p85α and FAM114A1 was determined via western blotting. f 293T cells were cotransfected with the indicated plasmids. Twenty-four hours after transfection, the cells were lysed and subjected to co-IP analysis. The samples were then analyzed via western blotting to detect interactions. g Py8119 cells stably expressing FAM114A1 were generated. The cells with (OE) or without (Vector) FAM114A1 overexpression were collected for co-IP analysis. The level of p110α that interacted with p85α was quantified after normalization to total p110α (right panel). h 293T cells were cotransfected with p85α and p110α plasmids. The cells were collected for co-IP after 24 h. The Co-IP samples from the beads were divided into 6 tubes and incubated with the indicated amount of the FAM114A1 recombinant protein for 2 h. The beads were washed thoroughly with lysis buffer, and the bound proteins were subjected to western blotting to detect p85α and p110α interactions. The data represent the means ± SEMs. P-values were determined by a two-tailed Student’s t -test ( b, g )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Immunopeptidomics, Expressing, Activation Assay, Knockdown, Isolation, Western Blot, Control, Immunohistochemistry, Co-Immunoprecipitation Assay, Transfection, Stable Transfection, Generated, Plasmid Preparation, Over Expression, Incubation, Recombinant, Lysis, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 binds E2F4 to inhibit its condensate formation. a Py8119 tumor cell lysates were collected and immunoprecipitated with anti-FAM114A1 (left panel), anti-E2F4 (right panel) antibodies, or IgG controls. The interaction between E2F4 and FAM114A1 was determined via western blotting. b , c Py8119 cells with (KD#1 and KD#2) and without (shCoo2) FAM114A1-knockdown were fixed and subjected to immunofluorescent (IF) staining with an anti-E2F4 antibody. DNA was visualized with DAPI ( b ). The percentage of cells with E2F4 condensates was quantified ( c ). Bar, 10 µm. d Py8119 cells were transfected with the E2F4-GFP plasmid. Twenty-four hours after transfection, the cells were fixed for confocal imaging. DNA was visualized with DAPI. Bar, 10 µm. e Py8119 cells were transfected with the indicated E2F4-GFP mutant plasmids. Twenty-four hours after transfection, the cells were fixed for confocal imaging. DNA was visualized with DAPI. Bar, 10 µm. f 293T cells were cotransfected with the FAM114A1-Myc plasmid together with the indicated E2F4-GFP mutants. The cells were collected for co-IP after 24 h. The samples were subjected to western blotting to determine interactions. g Samples from TNBC patients were subjected to IHC staining with an anti-FAM114A1 antibody or IF staining with an anti-E2F4 antibody. DNA was visualized with DAPI. The nuclear areas are highlighted with red dashed circles in inset #1. Representative E2F4 condensates are indicated by red arrows in inset #2. Bar, 10 µm. h FAM114A1 expression was quantified with ImageJ after IHC staining, and the samples were stratified into high- and low-FAM114A1 groups on the basis of median expression. The number of cells with E2F4 condensates was also quantified via IF staining. The data represent the means ± SEMs. P-values were determined via one-way ANOVA ( c ) or the χ 2 test ( h )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Immunoprecipitation, Western Blot, Knockdown, Staining, Transfection, Plasmid Preparation, Imaging, Mutagenesis, Co-Immunoprecipitation Assay, Immunohistochemistry, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: E2F4 condensates exhibit liquid-like properties and are suppressed by FAM114A1. a Droplet formation by E2F4-GFP was analyzed at the indicated concentrations with 500 mM NaCl at room temperature. E2F4-GFP (5 μM) was examined using droplet formation assays conducted at room temperature ( b , d ) or at 4 °C or 37 °C ( c ) with the indicated concentrations of NaCl ( b ) and with or without 5% Hex at 500 mM NaCl ( c ). For a – d , representative fluorescence and differential interference contrast (DIC) images of the droplets (left of each panel) and quantification of the size of the droplets (right of each panel) are shown. Each dot represents a droplet. Hex, 1,6-hexanediol; Bar, 20 µm. e Live-cell imaging of E2F4-GFP droplets. The arrows indicate representative fused E2F4 condensates. Bar, 10 µm. f E2F4-GFP droplets were subjected to a fluorescence recovery after photobleaching (FRAP) assay. The blue circle indicates the area for photobleaching, and the red circle indicates the area that served as a non-photobleaching control (left panel). The average values for the FRAP data are shown (right panel). Bar, 2 µm. g E2F4-GFP (5 μM) droplet formation was monitored in the absence or presence of the indicated concentrations of GST or GST-FAM114A1 recombinant proteins (left panel). The size of the droplets was quantified (right panel). Bar, 20 µm. The data represent the means and all the data points. The P-value was determined via one-way ANOVA ( g )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Fluorescence, Live Cell Imaging, Photobleaching Assay, Control, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 enhances E2F4-mediated MTDH expression. a Py8119 cells were subjected to IF staining with an anti-E2F4 antibody. DNA was visualized with DAPI. The cells and nuclei are highlighted with white and red dashed circles, respectively. Bar, 10 µm. b The fluorescence intensity of nuclear E2F4 in Py8119 (left panel) and TNBC patient samples (right panel) was quantified. n = 50 cells in each group. c , d Py8119-OVA-Luc cells with (KD#1 and KD#2) or without (shCoo2) stable FAM114A1-knockdown were used to extract cytosolic or nuclear proteins. The expression of E2F4 and FAM114A1 in the cytosol and nucleus was examined via western blotting ( c ). The cells were subjected to RNA and protein extraction. The mRNA and protein levels of FAM114A1 and MTDH were examined via RT‒qPCR and western blotting, respectively ( d ). e RNA sequencing data of TNBC patients from the TCGA dataset were extracted. The Spearman rank correlation between MTDH and FAM114A1 was plotted. n = 124 TNBC patients. f , g TNBC patient samples were employed for IHC staining with anti-FAM114A1 and anti-MTDH antibodies. Representative images are shown ( f ). The expression levels of FAM114A1 and MTDH were quantified with ImageJ and analyzed with Spearman’s rank correlation ( g ). n = 109 TNBC patients. Bar, 500 µm. The data represent the means ± SEMs. P-values were determined by two-tailed Student’s t -test ( b ) or one-way ANOVA ( d )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Expressing, Staining, Fluorescence, Knockdown, Western Blot, Protein Extraction, RNA Sequencing, Immunohistochemistry, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1-mediated immunosuppression is independent of its ability to regulate the cell cycle. a‒c Py8119-OVA-Luc tumor cells with FAM114A1-knockdown and the corresponding controls were used for the tumor sphere assay. Five days after culture, the spheres were collected for cell cycle and apoptotic analysis ( a ), and the proportion of cells in each phase and the proportion of apoptotic cells were quantified ( b , c ). n = 3 replicates per group. shCoo2: Py8119-OVA-Luc PLKO-shCoo2; KD#1: Py8119-OVA-Luc PLKO-shFAM114A1#1; KD#2: Py8119-OVA-Luc PLKO-shFAM114A1#2. d , e Py8119 tumor cells with and without FAM114A1-knockdown were orthotopically injected into nude mice. Tumor size and weight were evaluated at the endpoint ( d ). n = 7 and 8 mice for the control (shCoo2) and FAM114A1-knockdown (KD) groups, respectively. Tumors from the indicated groups were collected for IHC staining with anti-Ki67 and cleaved caspase 3 (CC-3) antibodies. The percentage of positive cells was determined ( d ). n = 5 tumors per group. f Py8119 cells were synchronized with a double thymidine block. The cells released at the indicated time points were subjected to flow cytometry analysis to examine the cell cycle distribution and antigen presentation status. asy. : asynchronized. g Cells at the indicated phases were subjected to immunofluorescence staining with an anti-E2F4 antibody. The percentage of cells with E2F4 condensates at the indicated phases was quantified (left panel). The antigen presentation status of cells at the indicated phases was quantified (right panel). h OT-I female mice were pretreated with 125 μg/mouse anti-CD8 antibody or the corresponding isotype control every two days for one week. Py8119-OVA-Luc tumor cells with/without FAM114A1-knockdown were orthotopically injected into the mice, and the mice were continually treated with 125 μg/mouse of anti-CD8 antibody or the corresponding isotype control twice per week until the end of the experiment. The tumor size was measured 6 weeks after tumor cell injection. Primary tumors were dissected and weighed at week 7. n = 6 mice for the FAM114A1 control (shCoo2)- and isotype (IgG)-treated groups; n = 8 mice for the FAM114A1-knockdown (KD)- and isotype (IgG)- or anti-CD8 antibody-treated groups. The data represent the means ± SEMs. P-values were determined via one-way ANOVA ( b , c , g , h ) or two-tailed Student’s t -test ( d , e )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Knockdown, Injection, Control, Immunohistochemistry, Blocking Assay, Flow Cytometry, Immunopeptidomics, Immunofluorescence, Staining, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Family with sequence similarity 114 member A1 orchestrates immune evasion in triple-negative breast cancer

doi: 10.1038/s41392-025-02472-9

Figure Lengend Snippet: FAM114A1 targeting sensitizes TNBC to ICB therapy. a , Schematic diagram of FAM114A1-inducible knockdown (iKD) combined with anti-PD-1 therapy in mouse models. b – d Py8119-OVA-Luc cells with inducible FAM114A1-knockdown were generated. The cells were orthotopically injected into the mammary fat pads of female OT-I mice. One week after the injection (when the tumors were established), the mice were administered doxycycline (iKD) and anti-PD-1 antibodies alone or in combination. Control mice received either an isotype control antibody (IgG) and/or vehicle (iNC). Tumor growth was monitored and measured weekly ( b ). The tumors were dissected at week 7, and representative tumors are shown ( c ). Tumor weight was measured ( d ). n = 12 mice per group. Bar, 1 cm. e – g Tumors from ( b ) were collected for IHC staining with an anti-CD8 antibody ( e ). The percentage of CD8 + T cells per field was quantified in each group ( f ). Tumors were digested, and flow cytometry assays were performed to examine the ratio of CD8 + T cells (CD3 + CD8 + ) to activated CD8 + T cells (CD8 + CD137 + ) in tumors ( g ). n = 6 tumors per group. Bar, 50 µm. h , i FAM114A1 expression was examined in the NCT02489448 immunotherapy TNBC patient cohort. The median expression level was used as the cutoff to define high- and low-FAM114A1 patients. The percentages of pathological complete response (pCR) and non-pCR patients are shown for both FAM114A1 -high and -low patients ( h ). The expression of FAM114A1 in pCR patients and non-pCR patients is shown ( i ). j Schematic diagram of the working model. The data represent the means ± SEMs. P-values were determined via one-way ANOVA ( b, d, f, g ), the χ 2 test ( h ), and two-tailed Student’s t -test ( i )

Article Snippet: pLKO plasmids encoding shRNAs targeting murine FAM114A1 (FAM114A1-KD#1, TRCN0000174803; and FAM114A1-KD#2, TRCN0000174459), murine E2f4 (E2F4-KD#1, TRCN0000348953; and E2F4-KD#2, TRCN0000331779), and murine B2m (B2m-KD#1, TRCN0000288438; and B2m-KD#2, TRCN0000295705) were purchased from Genewiz (Suzhou, China) and were cloned as described previously., For FAM114A1-inducible knockdown cell lines, the same two targets were used and cloned and inserted into the pTRIPZ vector (Addgene#127696).

Techniques: Knockdown, Generated, Injection, Control, Immunohistochemistry, Flow Cytometry, Expressing, Two Tailed Test