Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs decreases the ability of STING agonists to trigger an antiviral response. (A) T98G cells were treated with the 10 µM diABZI, 1 µM E7766, or 10 mg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP for 16 h prior to DAPI nuclear staining and image acquisition. Images are representative of three independent experiments. (B) As in A, except that cells were infected with the MPXV clade 2b strain S2626 for 48 h. Images are representative of three independent experiments. (C) T98G cells engineered to express control nontargeting or DNA-PKcs–targeting gRNA were treated with 10 µg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP at MOI 0.3 for 16 h, prior to DAPI nuclear staining and image acquisition. Graph shows the mean (±SEM, n = 3 independent experiments) percentage of infected (GFP + ) cells as measured by fluorescent microscopy. Statistical significance was assessed using two-tailed Student's t test. (D) Gating strategy for macrophages used in . (E) Histograms show the percentage of infected (GFP + ) cells as measured by flow cytometry, following treatment with STING agonists, in the presence or absence of NU7441, at two different MOIs. (F) Primary macrophages from healthy donors 1, 2, and 3 were pretreated with NU7441 prior to STING agonist treatment. Cells treated with a STING agonist, and they were set as 100% infection, and the effect of adding a NU7441 was assessed relative to this condition. Scale bars, 500 μm. **: P < 0.01; *: P < 0.05. Data are from at least three independent experiments. Related to .

Article Snippet: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

Techniques: Infection, Staining, Control, Microscopy, Two Tailed Test, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting the UFL1-AKT cascade suppresses triple-negative breast cancer progression

doi: 10.1038/s41467-026-68493-2

Figure Lengend Snippet: a , b TNBC patient-derived xenografts (PDXs) were subcutaneously implanted into nude mice. Saline, PDAU or NP (15 mg/kg) was injected intraperitoneally every 2 days when tumor volume reached 100 mm 3 . Mice were then treated with saline or cisplatin (5 mg/kg weekly, n = 6). Tumors were collected ( a ), and tumor weights were measured ( b ). Results represent the mean ± SD from six mice. c Cell lysates from PDXs treated with saline, PDAU or NP were subjected to immunoprecipitation with IgG, anti-UFL1 antibodies. The immunoprecipitates were blotted with the indicated antibodies. d Cell lysates from PDXs treated with saline, PDAU or NP were subjected to western blot with the indicated antibodies. e Representative images of H&E, Ki67 and cleaved PARP1 staining in xenograft models from ( a ). Scale bars, 50 μm. Data were analyzed by two-sided one-way ANOVA in ( b ). Source data are provided as a Source Data file.

Article Snippet: Anti-UFL1 (A303-456A, dilution: 1:1000) antibodies were purchased from BETHYL.

Techniques: Derivative Assay, Saline, Injection, Immunoprecipitation, Western Blot, Staining

Journal: Cancer Science

Article Title: Crucial Contribution of BACH1 to Bladder Cancer Progression via Upregulating Epithelial‐Mesenchymal Transition Pathway

doi: 10.1111/cas.70284

Figure Lengend Snippet: Relationship between BACH1 expression and prognosis of bladder cancer patients. (A) Immunohistochemical BACH1 staining of in BACH1‐positive and BACH1‐negative bladder cancer (BC). Original magnification, × 100. Scale bar, 100 μm (left panels) and × 400. Scale bar, 50 μm (right panels). (B, C) Kaplan–Meier plots of recurrence‐free (B) and cancer‐specific (C) survival rates for BACH1‐positive and BACH1negative BC patients. p values calculated by a log‐rank test are also indicated.

Article Snippet: The following antibodies were used: anti‐human BACH1 (1:1000, #A303‐058A; Bethyl Laboratories, Montgomery, TX, USA), anti‐mouse Bach1 (1:1000, #66762–1‐Ig; Proteintech), anti‐E‐cadherin (1:1000, #14472; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1:1000, #13116; Cell Signaling Technology), anti‐Vimentin (1:1000, #5741; Cell Signaling Technology), anti‐SNAI1 (1:1000, #13099–1‐AP; Proteintech), anti‐SNAI2 (1:1000, #12129–1‐AP; Proteintech), anti‐NF‐κB p65 (1:1000, #8242; Cell Signaling Technology), anti‐phospho NF‐κB p65 (pNF‐κB p65) (1:1000, #3033; Cell Signaling Technology), anti‐STAT3 (1:1000, #9139; Cell Signaling Technology), and anti‐phospho STAT3 (pSTAT3) (1:1000, #9145; Cell Signaling Technology).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Crucial Contribution of BACH1 to Bladder Cancer Progression via Upregulating Epithelial‐Mesenchymal Transition Pathway

doi: 10.1111/cas.70284

Figure Lengend Snippet: Molecular function of BACH1 in bladder cancer cell invasion, migration, and proliferation in vitro. (A,B) BACH1‐knockdown (siBACH1‐1 and siBACH1‐2) (A) and BACH1‐overexpressing (exBACH1) (B) RT4, UMUC3, UMUC13, T24, and 5637 cells were subjected to determine mRNA (left panels) and protein (right panels) levels of BACH1 by quantitative RT‐PCR and western blot analysis, respectively. Results are expressed as mean ± standard error of the mean (SEM) ( n = 3, left panels) or representative of three experiments (right panels). ** p < 0.01 and *** p < 0.001 by one‐way ANOVA with Steel multiple comparison test, compared with control siRNA‐introduced control (siCtrl) (A) or empty vector‐introduced control (EV) (B). (C, D) BACH1‐knockdown (C) and BACH1‐overexpressing (D) cell lines were subjected to transwell invasion assay with corresponding control cells (siCtrl or EV). Results are expressed as the mean ± SEM ( n = 4). * p < 0.05, ** p < 0.01 and *** p < 0.001 by one‐way ANOVA with Steel multiple comparison test, compared with siCtrl (C) or EV (D). Representative Diff‐Quick‐stained transwell membranes using 5637 cells are shown (C, D; left panels). Scale bar, 100 μm. (E, F) BACH1‐knockdown (E) and BACH1‐overexpressing (F) cell lines were subjected to wound healing assay with corresponding control cells (siCtrl or EV). Results are expressed as the mean ± SEM ( n = 6). *** p < 0.001 by one‐way ANOVA with Steel multiple comparison test, compared with siCtrl (E) or EV (F). Representative images of migrated 5637 cells are shown (E, F; left panels). Scale bar, 100 μm. (G) BACH1‐knockdown (upper panels) and BACH1‐overexpressing (lower panels) cell lines were subjected to proliferation assay with corresponding control cells (siCtri and EV). Results are expressed as the mean ± SEM ( n = 3). * p < 0.05 and *** p < 0.001 by one‐way ANOVA with Steel multiple comparison test, compared with siCtrl or EV at 72 h.

Article Snippet: The following antibodies were used: anti‐human BACH1 (1:1000, #A303‐058A; Bethyl Laboratories, Montgomery, TX, USA), anti‐mouse Bach1 (1:1000, #66762–1‐Ig; Proteintech), anti‐E‐cadherin (1:1000, #14472; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1:1000, #13116; Cell Signaling Technology), anti‐Vimentin (1:1000, #5741; Cell Signaling Technology), anti‐SNAI1 (1:1000, #13099–1‐AP; Proteintech), anti‐SNAI2 (1:1000, #12129–1‐AP; Proteintech), anti‐NF‐κB p65 (1:1000, #8242; Cell Signaling Technology), anti‐phospho NF‐κB p65 (pNF‐κB p65) (1:1000, #3033; Cell Signaling Technology), anti‐STAT3 (1:1000, #9139; Cell Signaling Technology), and anti‐phospho STAT3 (pSTAT3) (1:1000, #9145; Cell Signaling Technology).

Techniques: Migration, In Vitro, Knockdown, Quantitative RT-PCR, Western Blot, Comparison, Control, Plasmid Preparation, Transwell Invasion Assay, Diff-Quik, Staining, Wound Healing Assay, Proliferation Assay

Journal: Cancer Science

Article Title: Crucial Contribution of BACH1 to Bladder Cancer Progression via Upregulating Epithelial‐Mesenchymal Transition Pathway

doi: 10.1111/cas.70284

Figure Lengend Snippet: Gene Set Enrichment Analysis (GSEA) of BACH1‐knockdown bladder cancer cells. (A) Bubble plots showing the normalized enrichment scores (NES) of the 14 downregulated hallmark pathways with a false discovery rate (FDR) below 0.25 in BACH1‐knockdown UMUC3 cells compared with control siRNA‐transduced cells. (B) Enrichment patterns of hallmark TNF‐α signaling via NFκβ‐related, IL‐6/JAK/STAT3 signaling‐related, inflammatory response‐related, epithelial‐mesenchymal transition‐related, and hypoxia‐related gene sets determined by GSEA comparing BACH1‐knockdown and control cells. FDR‐adjusted p values ( q values) are also indicated.

Article Snippet: The following antibodies were used: anti‐human BACH1 (1:1000, #A303‐058A; Bethyl Laboratories, Montgomery, TX, USA), anti‐mouse Bach1 (1:1000, #66762–1‐Ig; Proteintech), anti‐E‐cadherin (1:1000, #14472; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1:1000, #13116; Cell Signaling Technology), anti‐Vimentin (1:1000, #5741; Cell Signaling Technology), anti‐SNAI1 (1:1000, #13099–1‐AP; Proteintech), anti‐SNAI2 (1:1000, #12129–1‐AP; Proteintech), anti‐NF‐κB p65 (1:1000, #8242; Cell Signaling Technology), anti‐phospho NF‐κB p65 (pNF‐κB p65) (1:1000, #3033; Cell Signaling Technology), anti‐STAT3 (1:1000, #9139; Cell Signaling Technology), and anti‐phospho STAT3 (pSTAT3) (1:1000, #9145; Cell Signaling Technology).

Techniques: Knockdown, Control

Journal: Cancer Science

Article Title: Crucial Contribution of BACH1 to Bladder Cancer Progression via Upregulating Epithelial‐Mesenchymal Transition Pathway

doi: 10.1111/cas.70284

Figure Lengend Snippet: Role of BACH1 in bladder cancer tumor progression and metastasis in vivo. (A) MB49 luc cells transduced with either a scrambled sgRNA construct (MB49 luc /Scramble) or Bach1‐KO construct (MB49 luc /Bach1‐KO) were subjected to determine the expression level of Bach1 by western blot analysis. (B) Estimated tumor volumes of MB49 luc /Scramble and MB49 luc /Bach1‐KO cell‐transplanted mice. Results are expressed as the mean ± standard error of the mean (SEM) ( n = 6). * p < 0.05 and *** p < 0.001 by Mann–Whitney U test, compared with MB49 luc /Bach1‐KO. (C) Photographic data (left panel) and weight (right panel) of excised MB49 luc /Scramble and MB49 luc /Bach1‐KO tumors at the endpoint. Scale bar, 5 mm (left panel). Results are expressed as the mean ± SEM ( n = 6). * p < 0.05 by Mann–Whitney U test, compared with MB49 luc /Bach1‐KO. (D) Top one tumor samples in (C) left panel were subjected to western blot analysis for E‐Cadherin, N‐Cadherin, Vimentin, SNAI1, SNAI2, Bach1, and β‐actin. (E, F) Bioluminescence imaging to assess fluorescence intensity of lungs in the in vivo metastasis assay. Representative photographs (E) and average fluorescence intensity (F) of lung tumors in MB49 luc /Scramble and MB49 luc /Bach1‐KO cell‐injected mice. Results are expressed as the mean ± SEM ( n = 3). * p < 0.05 by Mann–Whitney U test, compared with MB49 luc /Bach1‐KO. (G) Representative photographs of metastatic tumor nodules at the lung surface from mice injected with MB49 luc /Scramble (upper panel) and MB49 luc /Bach1‐KO (lower panel) cells. The yellow triangles indicate tumor nodules. Scale bar, 5 mm. (H) Representative H&E (upper panels) and immunohistochemical (IHC) Bach1 (lower panels) staining of lungs in (G). Original magnification × 100. Scale bar, 100 μm. (I) The number of metastatic nodules at the lung surface. Results are expressed as the mean ± SEM ( n = 8). ** P < 0.01 by Mann–Whitney U test, compared with MB49 luc /Bach1‐KO.

Article Snippet: The following antibodies were used: anti‐human BACH1 (1:1000, #A303‐058A; Bethyl Laboratories, Montgomery, TX, USA), anti‐mouse Bach1 (1:1000, #66762–1‐Ig; Proteintech), anti‐E‐cadherin (1:1000, #14472; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1:1000, #13116; Cell Signaling Technology), anti‐Vimentin (1:1000, #5741; Cell Signaling Technology), anti‐SNAI1 (1:1000, #13099–1‐AP; Proteintech), anti‐SNAI2 (1:1000, #12129–1‐AP; Proteintech), anti‐NF‐κB p65 (1:1000, #8242; Cell Signaling Technology), anti‐phospho NF‐κB p65 (pNF‐κB p65) (1:1000, #3033; Cell Signaling Technology), anti‐STAT3 (1:1000, #9139; Cell Signaling Technology), and anti‐phospho STAT3 (pSTAT3) (1:1000, #9145; Cell Signaling Technology).

Techniques: In Vivo, Transduction, Construct, Expressing, Western Blot, MANN-WHITNEY, Imaging, Fluorescence, Injection, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Crucial Contribution of BACH1 to Bladder Cancer Progression via Upregulating Epithelial‐Mesenchymal Transition Pathway

doi: 10.1111/cas.70284

Figure Lengend Snippet: BACH1 promotes cytokine signaling and immune modulation in bladder tumors. (A) Bubble plots showing the normalized enrichment scores (NES) of the top 15 upregulated hallmark pathways in tumors with high BACH1 mRNA expression compared to those with low BACH1 expression in The Cancer Genome Atlas Program (TCGA) datasets. (B) Enrichment patterns of hallmark gene sets related to TNF‐α signaling via NF‐κB and IL‐6/JAK/STAT3 signaling, as determined by Gene Set Enrichment Analysis comparing tumors with high versus low BACH1 expression. False discovery rate (FDR)‐adjusted p values ( q values) are also indicated. (C) BACH1‐knockdown (siBACH1‐1 and siBACH1‐2) (left panel) and BACH1‐overexpressing (exBACH1) (right panel) UMUC3 cells were subjected to determine mRNA levels of TNF and IL6 by quantitative RT‐PCR. Results are expressed as mean ± standard error of the mean (SEM) ( n = 3). ns: p > 0.05 by one‐way ANOVA with Steel multiple comparison test, compared with control siRNA‐introduced control (siCtrl) or empty vector‐introduced control (EV). (D) BACH1‐knockdown (left panel) and BACH1‐overexpressing (right panel) UMUC3 cells, along with their control cells, were subjected to determine protein expression of NF‐κB p65, phosphorylated NF‐κB p65 (pNF‐κB p65), STAT3, phosphorylated STAT3 (pSTAT3), and β‐actin by western blot analysis. Results are expressed as representative of three experiments. (E) MB49 luc cells transduced with either a scrambled sgRNA construct (MB49 luc /Scramble) or Bach1‐KO construct (MB49 luc /Bach1‐KO) tumor samples were subjected to quantitative RT‐PCR for Bach1 , Tnf , and Il6 . Results are expressed as mean ± SEM ( n = 6). *** p < 0.001 by one‐way ANOVA with Steel multiple comparison test for comparisons between MB49 luc /Scramble and MB49 luc /Bach1‐KO groups. (F) MB49 luc /Scramble and MB49 luc /Bach1‐KO tumor samples were subjected to western blot analysis for NF‐κB p65, pNF‐κB p65, STAT3, pSTAT3, Bach1, and β‐actin. Results are expressed as representative of three experiments.

Article Snippet: The following antibodies were used: anti‐human BACH1 (1:1000, #A303‐058A; Bethyl Laboratories, Montgomery, TX, USA), anti‐mouse Bach1 (1:1000, #66762–1‐Ig; Proteintech), anti‐E‐cadherin (1:1000, #14472; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1:1000, #13116; Cell Signaling Technology), anti‐Vimentin (1:1000, #5741; Cell Signaling Technology), anti‐SNAI1 (1:1000, #13099–1‐AP; Proteintech), anti‐SNAI2 (1:1000, #12129–1‐AP; Proteintech), anti‐NF‐κB p65 (1:1000, #8242; Cell Signaling Technology), anti‐phospho NF‐κB p65 (pNF‐κB p65) (1:1000, #3033; Cell Signaling Technology), anti‐STAT3 (1:1000, #9139; Cell Signaling Technology), and anti‐phospho STAT3 (pSTAT3) (1:1000, #9145; Cell Signaling Technology).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Comparison, Control, Plasmid Preparation, Western Blot, Transduction, Construct