Journal: Cancer letters

Article Title: DNA-PKcs promotes therapy resistance and metastatic recurrence in neuroblastoma

doi: 10.1016/j.canlet.2026.218383

Figure Lengend Snippet: (A, B) Correlation between PRKDC gene expression levels and NB tumor progression to stage 4. (C) Heatmap of differential expression of DNA replication and repair pathway (KEGG) genes in PTCs (green) at diagnosis and DTCs (red) at relapse. Color intensity represents expression level. Heatmap of KEGG pathways involved in DNA replication and repair in primary tumor cells (PTCs) at diagnosis (green) and in disseminated tumors cells (DTCs) at relapse (red). (D) PRKDC gene expression in PTCs at diagnosis, DTCs at diagnosis and in DTCs at relapse (Ambros study, n=86) revealed a significant upregulation of PRKDC in DTCs at relapse. (E) Western blot analysis in MYCN-amplified (BE(2)-C, LAN-1, IMR-32 and SK-N-DZ) and MYCN-non-amplified (SK-N-AS, SK-N-SH) NB cell lines (*p<0.05, ***p<0.001, ****p<0.0001).

Article Snippet: MYCN -amplified human NB cell lines (BE(2)-C (CVCL_V006), IMR-32 (CVCL_0346), LAN-1 (CVCL_1827) and SK-N-DZ (CVCL_1701)) and MYCN -non-amplified cells (SK-N-AS (CVCL_1700), and SK-N-SH (CVCL_0531)) were purchased from ATCC.

Techniques: Gene Expression, Quantitative Proteomics, Biomarker Discovery, Expressing, Western Blot, Amplification

Journal: Cancer letters

Article Title: DNA-PKcs promotes therapy resistance and metastatic recurrence in neuroblastoma

doi: 10.1016/j.canlet.2026.218383

Figure Lengend Snippet: (A, B) BE(2)-C and SK-N-DZ cells were treated with doxorubicin (A) or etoposide (B), either individually or in combination with peposertib. Clonogenic assay was performed to evaluate long-term cell viability of NB cells. BE(2)-C and SK-N-DZ cells were treated with peposertib (1000 nM), and doxorubicin (BE(2)-C, 75 nM; SK-N-DZ, 10 nM) and etoposide (BE(2)-C, 500 nM; SK-N-DZ, 100 nM). Drugs were removed and replaced with fresh media 48 h later, and colonies were allowed to grow for 14 days. (C, D) DNA damage following doxorubicin (C) or etoposide (D) therapy in combination with peposertib was evaluated in BE(2)C and SK-N-DZ using immunofluorescence staining of γ-H2AX (Ser139) (Red: γ-H2AX; Blue: DAPI; Green: Phalloidin). (E) Clonogenic assay was performed to evaluate long-term viability of NB cells. BE(2)-C NTC and PRKDC knockdown cells were treated with etoposide (500 nM) and doxorubicin (75 nM). Drugs were removed and replaced with fresh media 48 h later, and colonies were allowed to grow for 14 days; *p<0.05, ***p<0.001, ****p<0.0001.

Article Snippet: MYCN -amplified human NB cell lines (BE(2)-C (CVCL_V006), IMR-32 (CVCL_0346), LAN-1 (CVCL_1827) and SK-N-DZ (CVCL_1701)) and MYCN -non-amplified cells (SK-N-AS (CVCL_1700), and SK-N-SH (CVCL_0531)) were purchased from ATCC.

Techniques: Clonogenic Assay, Immunofluorescence, Staining, Knockdown

Journal: Cancer letters

Article Title: DNA-PKcs promotes therapy resistance and metastatic recurrence in neuroblastoma

doi: 10.1016/j.canlet.2026.218383

Figure Lengend Snippet: (A) BE(2)-C GFP-Luc cells were intravenously injected into NOD rag gamma mice. Chemotherapy commenced 6 days post-injection, with q.o.d doxorubicin (2.5 mg/kg, iv) and peposertib (100 mg/kg, gavage) as combination therapy, followed by a single dose of peposertib alone (100 mg/kg) the next day. Bioluminescent imaging (top) and GFP imaging (bottom) to demonstrate therapy outcomes on liver metastasis burden. (B) Bioluminescent signal quantification was used to measure liver metastasis burden at the end of the study (*p<0.05). (C) Gross photograph of GI track in doxorubicin and peposertib combination. (D) Mouse weight.

Article Snippet: MYCN -amplified human NB cell lines (BE(2)-C (CVCL_V006), IMR-32 (CVCL_0346), LAN-1 (CVCL_1827) and SK-N-DZ (CVCL_1701)) and MYCN -non-amplified cells (SK-N-AS (CVCL_1700), and SK-N-SH (CVCL_0531)) were purchased from ATCC.

Techniques: Injection, Imaging

Journal: Cancer letters

Article Title: DNA-PKcs promotes therapy resistance and metastatic recurrence in neuroblastoma

doi: 10.1016/j.canlet.2026.218383

Figure Lengend Snippet: (A) BE(2)-C cells were pre-treated with peposertib for 30 min before IR, then cells were irradiated at 1 Gy and peposertib was removed at different time points (top panel). BE(2)-C cells were irradiated at 1 Gy and peposertib was added at different time points post-IR (bottom panel). (B) Colony viability was measured by colony formation assay. (C) BE2C cells were cultured for 7 days to form colonies before initiating doxorubicin (75 and 150 nM) or IR (1 Gy and 4 Gy) therapy alone or in combination with peposertib. Following treatment, the media was refreshed 48 h later, and the colonies were permitted to grow for an additional 7 days. (D) Colony viability was measured by colony formation assay. (E) Similarly, SK-N-DZ cells were allowed to form colonies for 7 days prior to treatment with doxorubicin (10 or 20 nM) or ionizing radiation (0.5 Gy or 2 Gy), alone or in combination with peposertib. The culture medium was refreshed 48 h post-treatment, and colonies were incubated for an additional 7 days. (F) Represents the Colony viability measurement.; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: MYCN -amplified human NB cell lines (BE(2)-C (CVCL_V006), IMR-32 (CVCL_0346), LAN-1 (CVCL_1827) and SK-N-DZ (CVCL_1701)) and MYCN -non-amplified cells (SK-N-AS (CVCL_1700), and SK-N-SH (CVCL_0531)) were purchased from ATCC.

Techniques: Irradiation, Colony Assay, Cell Culture, Incubation

Journal: Cancer letters

Article Title: DNA-PKcs promotes therapy resistance and metastatic recurrence in neuroblastoma

doi: 10.1016/j.canlet.2026.218383

Figure Lengend Snippet: (A) Area of irradiation outlined by a light window. (B) BE(2)-C GFP-Luc cells were injected iv into NOD rag gamma mice. Radiation therapy started 6 days after cancer cell injection with daily 2 Gy irradiation alone or in combination with peposertib (gavage; 100 mg/kg) for 4 days. (C) Bioluminescent imaging and (D) GFP imaging demonstrate therapy outcomes on liver metastasis burden. (E) Bioluminescent signal quantification to measure liver metastasis burden at the end of the study (*p < 0.05). (F) Mouse weight. (G) Gross photograph of liver metastasis. (H) H&E imaging of liver metastasis and normal liver adjacent to metastatic tumors. (I ) DNA-PKcs expression in surviving colonies after acute (5 Gy) and chronic (1 Gy × 5 times) IR exposure. (J ) BE(2)-C cells subjected to repeated low-dose irradiation (1 Gy × 5) exhibited increased resistance to acute high-dose radiotherapy (5 Gy) relative to the parental cell line. (K ) PRKDC gene expression in NB tumors with recurrence or progression (Versteeg study, n = 87) revealed a significant upregulation of PRKDC in tumors at relapse. (L) Side-by-side UMAP visualization of NB tumor cell subclusters in post-treatment neuroblastoma at primary (n = 21,248 cells) and metastatic (n = 24,526 cells) sites after re-clustering. Colors represent assigned cell types within NB tumors. The dot plot shows PRKDC gene expression in tumor subpopulations in NB at primary and metastatic locations post-induction treatment. The color scale represents scaled average expression of PRKDC in each tumor type, and the size of the circle indicates the proportion of cells expressing PRKDC . (M) DNA-PKcs inhibition in combination with radiotherapy disrupts self-renewal capacity of BE(2)-C cells. BE(2)-C cells were treated with 5 cycles of doxorubicin (50 nM) therapy or irradiation (1 Gy) alone or in combination with peposertib. (N) Elevated DNA-PKcs expression in metastatic NB cells confers a survival advantage under chemotherapeutic and radiotherapeutic stress. DNA-PKcs inhibition combined with repeated low-dose ionizing radiation impairs cancer cell self-renewal capacity and suppresses tumor relapse.

Article Snippet: MYCN -amplified human NB cell lines (BE(2)-C (CVCL_V006), IMR-32 (CVCL_0346), LAN-1 (CVCL_1827) and SK-N-DZ (CVCL_1701)) and MYCN -non-amplified cells (SK-N-AS (CVCL_1700), and SK-N-SH (CVCL_0531)) were purchased from ATCC.

Techniques: Irradiation, Injection, Imaging, Expressing, Gene Expression, Inhibition

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 inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

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 selectively counteracts CDNs. (A) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of c-di-GMP. Graph presents the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by one-way ANOVA. ns, not significant. (B) T98G cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (C) As in B, except that gene expression analyses were conducted. Graphs present the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by two-tailed Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05. (D) Experimental scheme for human primary monocyte isolation and treatment . Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. (E) Flow cytometry analysis of macrophages prepared as in . (F) Gene expression analyses were performed on human primary cells treated as described in . Graphs present the mean (±SEM) expression of IFIT1 in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (H) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (J) Control and DNA-PKcs knockout THP-1 cells were treated with 1 µM E7766 for 6 h prior to gene expression analysis. Graphs present mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) Control and DNA-PKcs knockout THP-1 cells were treated with 10 µM diABZI for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05. Related to . Source data are available for this figure: .

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: In Vitro, Gene Expression, Two Tailed Test, Isolation, Flow Cytometry, Expressing, Control, Knock-Out

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 inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Article Snippet: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

Techniques: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

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 selectively counteracts CDNs. (A) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of c-di-GMP. Graph presents the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by one-way ANOVA. ns, not significant. (B) T98G cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (C) As in B, except that gene expression analyses were conducted. Graphs present the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by two-tailed Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05. (D) Experimental scheme for human primary monocyte isolation and treatment . Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. (E) Flow cytometry analysis of macrophages prepared as in . (F) Gene expression analyses were performed on human primary cells treated as described in . Graphs present the mean (±SEM) expression of IFIT1 in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (H) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (J) Control and DNA-PKcs knockout THP-1 cells were treated with 1 µM E7766 for 6 h prior to gene expression analysis. Graphs present mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) Control and DNA-PKcs knockout THP-1 cells were treated with 10 µM diABZI for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05. Related to . Source data are available for this figure: .

Article Snippet: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

Techniques: In Vitro, Gene Expression, Two Tailed Test, Isolation, Flow Cytometry, Expressing, Control, Knock-Out

Journal: Journal of Cell Communication and Signaling

Article Title: Human brain pericytes protect the blood–brain barrier from triple‐negative breast cancer cells while promoting tumor aggressiveness

doi: 10.1002/ccs3.70070

Figure Lengend Snippet: Brain pericyte secretions mediate the inhibition of TNBC cell adhesion to the BBB. (A) Experimental workflow for adhesion assay. All BBB experiments were performed using ECGMV2 medium with supplements for all conditions from day −6 to the end of the experiment. The astrocyte medium was renewed 24 h before the adhesion experiment with the same ECGMV2 medium. Endothelial cells (ECs) and brain pericytes (hBPs) were co‐cultured for 5 days, and inserts containing brain‐like endothelial cells (hBLECs) were then either maintained with hBPs or transferred to wells without hBPs. After 24 hours, and prior to MDA‐MB‐231 cells seeding, inserts were subjected to the following conditions: maintained without hBPs (hBLEC 24h); transferred to hBPs previously cultured alone for 24 hours (hBLEC 24h then hBP); transferred to wells without hBPs but containing either culture medium (hBLEC) or CM from co‐culture (hBLEC + CM coc ); transferred to wells with astrocytes (hBLEC + A) or maintained in co‐culture with hBPs (hBLEC + hBP). (B–G), Quantification (B, D, F) of MDA‐MB‐231 cell adhesion to hBLECs after 3 h of incubation and representative images (C, E, G) of adhered MDA‐MB‐231 cells (green) under the different conditions. Data are obtained from three independent experiments, with three technical replicates per condition. Statistical analyses were performed using one‐way ANOVA followed by Tukey's test (B), Welch's ANOVA followed by Dunnett's T3 test (D) and Kruskal–Wallis test followed by Dunn's test (F). CM coc = conditioned medium from 24 h hBLECs and hBPs coculture, A = astrocytes, hBP mono = 24 h hBPs monoculture. Scale bar = 750 µm. BBB, blood–brain barrier; ECGMV2, EC Growth Medium MV 2; ECs, endothelial cells; hBLECs, human brain‐like endothelial cells; ns, non‐significant; hBPs, human brain pericytes; TNBC, triple‐negative breast cancer. **** p ≤ 0.0001; * p ≤ 0.05.

Article Snippet: Differentiated ECs were cultured in 100‐mm Petri dishes (Corning) coated with gelatin (Sigma‐Aldrich), in EC growth medium MV 2 (ECGMV2 + Supplement mix C‐39226, PromoCell) and gentamicin (50 μg/ml; Biowest). hBPs (female) were provided by Dr. Fumitaka Shimizu and Pr.

Techniques: Inhibition, Cell Adhesion Assay, Cell Culture, Co-Culture Assay, Incubation

Journal: Journal of Cell Communication and Signaling

Article Title: Human brain pericytes protect the blood–brain barrier from triple‐negative breast cancer cells while promoting tumor aggressiveness

doi: 10.1002/ccs3.70070

Figure Lengend Snippet: Brain pericytes limit TNBC cell adhesion to the BBB under hypoxic conditions. (A) Experimental workflow for adhesion assay in hypoxic conditions. All BBB experiments were performed using ECGMV2 medium with supplements for all conditions from day −6 to the end of the experiment. After 5 days of co‐culture with brain pericytes (hBPs), inserts containing brain‐like endothelial cells (hBLECs) were exposed to hypoxia for 24 h in the presence or absence of hBPs. Parallel conditions maintained in normoxia served as controls. (B–E) Quantification (B, D) of MDA‐MB‐231 cell adhesion to hBLECs after 3 h of incubation and representative images (C, E) of adhered MDA‐MB‐231 cells (green) under the different conditions. Data are obtained from three independent experiments, with three technical replicates per condition. Statistical analyses were performed using Welch's ANOVA followed by Dunnett's T3 test (B) and Kruskal‐Wallis test followed by Dunn's test (D). hBP‐CM mono = conditioned medium from 24 h hBPs monoculture. Scale bar = 750 μm. BBB, blood–brain barrier; ECGMV2, EC Growth Medium MV 2; hBLECs, human brain‐like endothelial cells; hBPs, human brain pericytes; ns, non‐significant; TNBC, triple‐negative breast cancer. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

Article Snippet: Differentiated ECs were cultured in 100‐mm Petri dishes (Corning) coated with gelatin (Sigma‐Aldrich), in EC growth medium MV 2 (ECGMV2 + Supplement mix C‐39226, PromoCell) and gentamicin (50 μg/ml; Biowest). hBPs (female) were provided by Dr. Fumitaka Shimizu and Pr.

Techniques: Cell Adhesion Assay, Co-Culture Assay, Incubation

Journal: Journal of Cell Communication and Signaling

Article Title: Human brain pericytes protect the blood–brain barrier from triple‐negative breast cancer cells while promoting tumor aggressiveness

doi: 10.1002/ccs3.70070

Figure Lengend Snippet: Brain pericytes maintain BBB integrity in the presence of TNBC cells under hypoxic conditions and supplement deprivation. (A) After 5 days of co‐culture with brain pericytes (hBPs) in ECGMV2 medium with supplements, inserts containing brain‐like endothelial cells (hBLECs) co‐cultured with hBPs were exposed to hypoxia for 16 h following medium replacement with ECGMV2 basal medium (without supplements). Then, endothelial permeability (Pe) to Lucifer Yellow was assessed after 3 h of incubation with MDA‐MB‐231 cells, in the presence or absence of hBPs, under normoxic and hypoxic conditions. (B) Permeability values (expressed in x10 −3 cm/min ± SD) are summarized in a table. (C) Representative images of endothelial Claudin‐5 (magenta) immunostaining in the absence or presence of hBPs after 3 h of incubation with MDA‐MB‐231 cells (green) under hypoxic conditions in basal medium. Data are obtained from three independent experiments, with three technical replicates per condition. Statistical analyses were performed using Welch's ANOVA followed by Dunnett's T3 test. MDA = MDA‐MB‐231 cells. Scale bars = 300 μm (10X) and 100 μm (40X). BBB, blood–brain barrier; ECGMV2, EC Growth Medium MV 2; hBLECs, human brain‐like endothelial cells; hBPs, human brain pericytes; ns, non‐significant; TNBC, triple‐negative breast cancer. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

Article Snippet: Differentiated ECs were cultured in 100‐mm Petri dishes (Corning) coated with gelatin (Sigma‐Aldrich), in EC growth medium MV 2 (ECGMV2 + Supplement mix C‐39226, PromoCell) and gentamicin (50 μg/ml; Biowest). hBPs (female) were provided by Dr. Fumitaka Shimizu and Pr.

Techniques: Co-Culture Assay, Cell Culture, Permeability, Incubation, Immunostaining

Journal: Journal of Advanced Research

Article Title: HDAC2 enhances the antimicrobial activity of neutrophils by promoting the formation of neutrophil extracellular traps (NETs) in sepsis

doi: 10.1016/j.jare.2025.08.041

Figure Lengend Snippet: HDAC2 enhances antimicrobial activities against E.coli in mice. (A) Bacterial loads in the blood were calculated in HDAC2-knockout and wild type mice. (A)The picture of the bacterial clones was taken by BIO-RAD ChemiDoc“MP Imaging System. (B)The number of colony-forming units (CFU) in the blood of mice was calculated. HDAC2 WT and HDAC2 KO mice were administrated with E. coli- GFP (5 × 10 7 ) by intravenous Injection. Blood (20 μl) was taken from the tail vein of the mice, applied to the pretreated solid MHA medium, and incubated at 37 °C for 20 h. Count the colonies on the culture medium and take pictures. The experiments were performed in quintuplicate, data are presented as the means ± SEM. of independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t -test). (B) Bacterial loads in the kidneys were calculated in HDAC2-knockout and wild type mice. The picture of kidneys from HDAC2 WT and HDAC2 KO mice were taken. The number of colony-forming units (CFU) in the kidneys of mice was calculated. The kidneys from HDAC2 KO and HDAC2 WT mice were weighed, ground, spread on the MHA plates, incubated for 16–24 h at 37 °C, and then bacterial clones were counted. The experiments were performed in quintuplicate, data are presented as the means ± SEM. of independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t -test). n = 5 mice per group. (C) H&E staining of livers and kidneys. The arrows indicate damage section of tissue. (D) Serum CRP and PCT levels of mice with E. coli- GFP bacteremia at 24 h. The CRP and PCT were measured by ELISA. Data are shown as mean ± SEM. *p < 0.05; n = 5 mice per group. ns, not significant.

Article Snippet: Concentrations of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), histone deacetylase 2 (HDAC2), PCT, CRP and myeloperoxidase (MPO) in mouse serum were quantified using their respective enzyme-linked immunosorbent assay (ELISA) kits, following the protocols provided by the manufacturer (Boster Biological Technology, Wuhan, China).

Techniques: Knock-Out, Clone Assay, Imaging, Injection, Incubation, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay