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hoechst 33258  (Abcam)


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    Structured Review

    Abcam hoechst 33258

    Hoechst 33258, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 490 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ab91335/Cell+Counting+Kit+8+(WST-8+%2F+CCK8)+-+1000test/pmc10484973-24-0-5
    Average 99 stars, based on 490 article reviews
    hoechst 33258 - by Bioz Stars, 2026-09
    99/100 stars

    Images

    1) Product Images from "CD82 expression marks the endothelium to hematopoietic transition at the onset of blood specification in human"

    Article Title: CD82 expression marks the endothelium to hematopoietic transition at the onset of blood specification in human

    Journal: iScience

    doi: 10.1016/j.isci.2023.107583


    Figure Legend Snippet:

    Techniques Used: Recombinant, Derivative Assay, Cell Culture, Flow Cytometry

    Related Articles

    Labeling:

    Article Title: Ferroptosis inhibition protects vascular endothelial cells and maintains integrity of the blood-spinal cord barrier after spinal cord injury
    Article Snippet: Samples were incubated with the following secondary antibodies for 1 hour at room temperature: Alexa Fluor 555-labeled goat anti-mouse IgG (H+L) (1:500; Abcam, Cambridge, MA, USA, Cat# ab150118, RRID: AB_2714033) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) (1:500; Beyotime Biotechnology, Shanghai, China, Cat# A0423; RRID: AB_2891323). .. Nuclei were labeled using 4′,6-diamine-2-benzene (DAPI) (Abcam, Cat# ab104139) or Hoechst 33342 (Abcam, Cat# ab228551). .. Samples were examined using laser confocal microscopy (LSM 980; Carl Zeiss, Oberkochen, Germany).

    Staining:

    Article Title: Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury
    Article Snippet: Next, corresponding secondary antibodies were applied at 20–24°C for 2 hours (Alexa Flour 488-conjugated AffiniPure Goat Anti-Mouse IgG(H+L), 1:800, Jackson ImmunoResearch Laboratories, West Grove, PA, USA, Cat# 115-545-003, RRID: AB_2338840; Alexa Flour 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L), 1:800, Jackson ImmunoResearch Laboratories, Cat# 111-545-003, RRID:AB_2338046; Alexa Flour Cy3-conjugated AffiniPure Goat Anti-Mouse IgG(H+L), 1:800, Jackson ImmunoResearch Laboratories, Cat# 115-165-003, RRID: AB_2338680; Alexa Flour Cy3-conjugated AffiniPure Goat Anti-Rat IgG(H+L), 1:800, Jackson ImmunoResearch Laboratories, Cat# 112-165-003, RRID: AB_2338240; Alexa Flour Cy3-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L), 1:800, Jackson ImmunoResearch Laboratories, Cat# 111-165-003, RRID: AB_2338240; and Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L), 1:800, Jackson ImmunoResearch Laboratories, Cat# 111-605-003, RRID: AB_2338072). .. Nuclei were stained with Hoechst 33342 (1:2000, Cat# ab228551, Abcam) for 15 minutes. .. Images were obtained on a Zeiss (Oberkochen, Germany) fluorescence microscope and fluorescence intensity was analyzed by ImageJ.

    Growth Inhibition Assay:

    Article Title: Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor
    Article Snippet: .. Cell growth inhibition assay was assessed with CCK8 kit (Abcam, UK, ab228554) by a microplate reader after 48 h of exposure to a serial dilution of COH29 (0.5–100 μM). .. The half-maximal inhibitory concentration (IC50) of COH29 in BT12, Re1P6, and CHLA266 cells was analyzed using AT Bioquest tool ( https://www.aatbio.com ).

    Serial Dilution:

    Article Title: Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor
    Article Snippet: .. Cell growth inhibition assay was assessed with CCK8 kit (Abcam, UK, ab228554) by a microplate reader after 48 h of exposure to a serial dilution of COH29 (0.5–100 μM). .. The half-maximal inhibitory concentration (IC50) of COH29 in BT12, Re1P6, and CHLA266 cells was analyzed using AT Bioquest tool ( https://www.aatbio.com ).

    Microscopy:

    Article Title: Astragaloside IV restrains pyroptosis and fibrotic development of pulmonary artery smooth muscle cells to ameliorate pulmonary artery hypertension through the PHD2/HIF1α signaling pathway
    Article Snippet: The sections were firstly permeabilized with 0.1% Triton X-100 for 30 min, and blocked with 5% bovine serum albumin for 1 h. Then, the sections were incubated overnight with primary antibody against NLRP3 (Abcam, Cambridge, UK, #ab263899), PHD2 (Abcam, #ab226890) and HIF1α (Abcam, #ab179483) at 4 °C, and incubated with Alexa Fluor®488 labeled IgG secondary antibody (Abcam, #ab150077). .. The nuclei were counterstained with Hoechst 33,258 (Abcam, #ab228550), and the image was visualized under a microscope and analyzed by the image J software. ..

    Software:

    Article Title: Astragaloside IV restrains pyroptosis and fibrotic development of pulmonary artery smooth muscle cells to ameliorate pulmonary artery hypertension through the PHD2/HIF1α signaling pathway
    Article Snippet: The sections were firstly permeabilized with 0.1% Triton X-100 for 30 min, and blocked with 5% bovine serum albumin for 1 h. Then, the sections were incubated overnight with primary antibody against NLRP3 (Abcam, Cambridge, UK, #ab263899), PHD2 (Abcam, #ab226890) and HIF1α (Abcam, #ab179483) at 4 °C, and incubated with Alexa Fluor®488 labeled IgG secondary antibody (Abcam, #ab150077). .. The nuclei were counterstained with Hoechst 33,258 (Abcam, #ab228550), and the image was visualized under a microscope and analyzed by the image J software. ..

    CCK-8 Assay:

    Article Title: A novel combination therapy with Cabozantinib and Honokiol effectively inhibits c-Met-Nrf2-induced renal tumor growth through increased oxidative stress
    Article Snippet: Anti-β-actin (Cat. No. #A2228) was obtained from Sigma-Aldrich (Burlington, MA). .. Anti-Rubicon (Cat No. #Ab92388) and the CCK-8 kit were purchased from Abcam (Waltham, MA). .. Anti-Sp1 (Cat. No. #14027) was obtained from Santa Cruz Biotechnology (Dallas, TX), and anti-GAPDH (Cat. No. #A5028) was purchased from Selleckchem.

    other:

    Article Title: Low-dose daylight exposure induces nitric oxide release and maintains cell viability in vitro
    Article Snippet: Assessment of cell survival after light exposure was performed using a cell counting kit 8 (WST-8, Abcam ab228554).

    Cell Culture:

    Article Title: Biocompatible Nanostructured Silver-Incorporated Implant Surfaces Show Effective Antibacterial, Osteogenic, and Anti-Inflammatory Effects in vitro and in Rat Model
    Article Snippet: .. At the designated time points, the media were changed to 500 μL fresh CCK8 medium (GM or OM with 10% CCK-8, ab228554, Abcam, Netherlands), and the cells were cultured for an additional 2 h. The CCK8 medium was transferred to a new 96-well plate (100 μL/well), and the absorbance was read at 450 nm. ..

    Incubation:

    Article Title: PCBP2 as an intrinsic aging factor regulates the senescence of hBMSCs through the ROS-FGF2 signaling axis
    Article Snippet: .. Then, 10 μL of CCK-8 reagent (WST-8/CCK8, ab228554, Abcam, USA) and 90 μL of serum-free medium were added to each well, and incubated for 2 hours in the 37°C, 5% CO2 and humid incubator. ..



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    Image Search Results


    Journal: iScience

    Article Title: CD82 expression marks the endothelium to hematopoietic transition at the onset of blood specification in human

    doi: 10.1016/j.isci.2023.107583

    Figure Lengend Snippet:

    Article Snippet: Hoechst 33258 (1/100) (1/100) , Abcam , Ab228550.

    Techniques: Recombinant, Derivative Assay, Cell Culture, Flow Cytometry

    TGFBR1 and BMPR2 are the main effector targets of miR-30-3p in HNSCC. ( a ) Predicted interaction between target proteins was analyzed using STRING database. ( b ) Heatmap representing predicted target gene expression was determined by RT-qPCR in HNSCC cell lines ( n = 4–7). ( c ) Western blot analysis of CAL27, CAL33, and SCC9 cell lines overexpressing miR-30a-3p or miR-30e-3p (c for miR-ctrl, a for miR-30a-3p and e for miR-30e-3p, n = 3–12, * p < 0.05, ** p < 0.01 and *** p < 0.001). ( d ) Immunofluorescence analysis by confocal microscopy of HNSCC cell lines overexpressing miR-30a-3p or miR-30e-3p ( n = 3–4). ( e ) Predicted pairing region between miR-30a-3p or miR-30e-3p and TGFBR1 (position 812–818) and BMPR2 (position 1411–1417). ( f ) Evasion of CAL27, CAL33, and SCC9 cell lines with TGF-β or BMP inhibitors ( n = 16–49 spheroids, * p < 0.05, ** p < 0.01 and *** p < 0.001). ( g ) Survival fraction of CAL27, CAL33, and SCC9 cell lines with TGF-β or BMP inhibitors ( n = 9–12, * p < 0.05 and *** p < 0.001).

    Journal: International Journal of Molecular Sciences

    Article Title: Tumor-Suppressive and Immunomodulating Activity of miR-30a-3p and miR-30e-3p in HNSCC Cells and Tumoroids

    doi: 10.3390/ijms241311178

    Figure Lengend Snippet: TGFBR1 and BMPR2 are the main effector targets of miR-30-3p in HNSCC. ( a ) Predicted interaction between target proteins was analyzed using STRING database. ( b ) Heatmap representing predicted target gene expression was determined by RT-qPCR in HNSCC cell lines ( n = 4–7). ( c ) Western blot analysis of CAL27, CAL33, and SCC9 cell lines overexpressing miR-30a-3p or miR-30e-3p (c for miR-ctrl, a for miR-30a-3p and e for miR-30e-3p, n = 3–12, * p < 0.05, ** p < 0.01 and *** p < 0.001). ( d ) Immunofluorescence analysis by confocal microscopy of HNSCC cell lines overexpressing miR-30a-3p or miR-30e-3p ( n = 3–4). ( e ) Predicted pairing region between miR-30a-3p or miR-30e-3p and TGFBR1 (position 812–818) and BMPR2 (position 1411–1417). ( f ) Evasion of CAL27, CAL33, and SCC9 cell lines with TGF-β or BMP inhibitors ( n = 16–49 spheroids, * p < 0.05, ** p < 0.01 and *** p < 0.001). ( g ) Survival fraction of CAL27, CAL33, and SCC9 cell lines with TGF-β or BMP inhibitors ( n = 9–12, * p < 0.05 and *** p < 0.001).

    Article Snippet: Samples were blocked in PBS/5% BSA/0.3% Triton X-100 for 1 h and incubated overnight at 4 °C with TGFBR1 (ab235178, 1/100, Abcam) or BMPR2 (ab130206, 1/100, Abcam).

    Techniques: Targeted Gene Expression, Quantitative RT-PCR, Western Blot, Immunofluorescence, Confocal Microscopy

    Overexpression of miR-30-3p slows down HNSCC patient-derived tumoroid growth and development. ( a ) Immunohistochemical staining of Keratin 34BE12, p40, p63, and hematoxylin and eosin staining in tumoroid versus the original tumor. ( b ) miR-30a-3p and miR-30e-3p expression was determined by RT-qPCR in tumoroids ( n = 5, *** p < 0.001). ( c ) miR-30a-3p and miR-30e-3p expression was determined by RT-qPCR in tumoroids post-transfection ( n = 4–5, * p < 0.05) ( d ) Number of tumoroids remaining at day 6 post-transfection with mir-30a-3p or miR-30e-3p ( n = 10–20 tumoroids, *** p < 0.001). ( e ) Volume of tumoroids at day 6 transfected with miR-30a-3p or miR-30e-3p. Estimated volume = (4/3) × π (d1/2 × d2/2 × d3/3) ( n = 10–20 tumoroids, * p < 0.05 and ** p < 0.01). ( f ) TGFBR1 and BMPR2 expressions were determined by RT-qPCR in tumoroids ( n = 4–6, ** p < 0.01 and *** p < 0.001). ( g ) BMPR2 expressions were determined by immunostaining in tumoroids ( n = 3).

    Journal: International Journal of Molecular Sciences

    Article Title: Tumor-Suppressive and Immunomodulating Activity of miR-30a-3p and miR-30e-3p in HNSCC Cells and Tumoroids

    doi: 10.3390/ijms241311178

    Figure Lengend Snippet: Overexpression of miR-30-3p slows down HNSCC patient-derived tumoroid growth and development. ( a ) Immunohistochemical staining of Keratin 34BE12, p40, p63, and hematoxylin and eosin staining in tumoroid versus the original tumor. ( b ) miR-30a-3p and miR-30e-3p expression was determined by RT-qPCR in tumoroids ( n = 5, *** p < 0.001). ( c ) miR-30a-3p and miR-30e-3p expression was determined by RT-qPCR in tumoroids post-transfection ( n = 4–5, * p < 0.05) ( d ) Number of tumoroids remaining at day 6 post-transfection with mir-30a-3p or miR-30e-3p ( n = 10–20 tumoroids, *** p < 0.001). ( e ) Volume of tumoroids at day 6 transfected with miR-30a-3p or miR-30e-3p. Estimated volume = (4/3) × π (d1/2 × d2/2 × d3/3) ( n = 10–20 tumoroids, * p < 0.05 and ** p < 0.01). ( f ) TGFBR1 and BMPR2 expressions were determined by RT-qPCR in tumoroids ( n = 4–6, ** p < 0.01 and *** p < 0.001). ( g ) BMPR2 expressions were determined by immunostaining in tumoroids ( n = 3).

    Article Snippet: Samples were blocked in PBS/5% BSA/0.3% Triton X-100 for 1 h and incubated overnight at 4 °C with TGFBR1 (ab235178, 1/100, Abcam) or BMPR2 (ab130206, 1/100, Abcam).

    Techniques: Over Expression, Derivative Assay, Immunohistochemical staining, Staining, Expressing, Quantitative RT-PCR, Transfection, Immunostaining

    miR-30a-3p and miR-30e-3p influence macrophages’ immune response to HNSCC cells. ( a ) Images of M0-polarized THP-1 exposed to CAL27, CAL33, SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media in phase-contrast microscopy (×10). ( b ) Western blot analysis of CD86 in M0-polarized THP-1 exposed to CAL27, CAL33, and SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media ( n = 7–10, * p < 0.05, ** p < 0.01, *** p < 0.001). ( c ) TGFBR1 expression was determined by RT-qPCR in M0-polarized THP-1 exposed to CAL27, CAL33, SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media ( n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001). ( d ) Western blot analysis of CD163 in M0-polarized THP-1 exposed to CAL27, CAL33, and SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media ( n = 8–10, * p < 0.05, ** p < 0.01, *** p < 0.001). ( e ) Images of MO-polarized THP-1 cells (green) phagocytosing miR-30a-3p or miR-30e-3p-transfected HNSCC cells (red) in confocal microscopy 20× and 63×. ( f ) Percentage of phagocytosis, calculated as the number of M0-polarized THP-1 cells phagocytosing transfected HNSCC cells/total number of M0-polarized THP-1 × 100 ( n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001). ( g ) Percentage of phagocytosis with antibody antiCD47, calculated as the number of M0-polarized THP-1 cells phagocytosing transfected HNSCC cells/total number of M0-polarized THP-1 × 100 ( n = 10–18, * p < 0.05).

    Journal: International Journal of Molecular Sciences

    Article Title: Tumor-Suppressive and Immunomodulating Activity of miR-30a-3p and miR-30e-3p in HNSCC Cells and Tumoroids

    doi: 10.3390/ijms241311178

    Figure Lengend Snippet: miR-30a-3p and miR-30e-3p influence macrophages’ immune response to HNSCC cells. ( a ) Images of M0-polarized THP-1 exposed to CAL27, CAL33, SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media in phase-contrast microscopy (×10). ( b ) Western blot analysis of CD86 in M0-polarized THP-1 exposed to CAL27, CAL33, and SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media ( n = 7–10, * p < 0.05, ** p < 0.01, *** p < 0.001). ( c ) TGFBR1 expression was determined by RT-qPCR in M0-polarized THP-1 exposed to CAL27, CAL33, SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media ( n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001). ( d ) Western blot analysis of CD163 in M0-polarized THP-1 exposed to CAL27, CAL33, and SCC9 overexpressing miR-30a-3p and miR-30e-3p conditioned media ( n = 8–10, * p < 0.05, ** p < 0.01, *** p < 0.001). ( e ) Images of MO-polarized THP-1 cells (green) phagocytosing miR-30a-3p or miR-30e-3p-transfected HNSCC cells (red) in confocal microscopy 20× and 63×. ( f ) Percentage of phagocytosis, calculated as the number of M0-polarized THP-1 cells phagocytosing transfected HNSCC cells/total number of M0-polarized THP-1 × 100 ( n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001). ( g ) Percentage of phagocytosis with antibody antiCD47, calculated as the number of M0-polarized THP-1 cells phagocytosing transfected HNSCC cells/total number of M0-polarized THP-1 × 100 ( n = 10–18, * p < 0.05).

    Article Snippet: Samples were blocked in PBS/5% BSA/0.3% Triton X-100 for 1 h and incubated overnight at 4 °C with TGFBR1 (ab235178, 1/100, Abcam) or BMPR2 (ab130206, 1/100, Abcam).

    Techniques: Microscopy, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Confocal Microscopy

    (A) Kaplan-Meier survival plots of TNBC breast cancer patients with low or high PFKFB4 signature score in TCGA (n = 149) and GEO: GSE21653 (n = 85) database. Medium cut point of PFKFB4 signature score was used to stratified patients. p values were calculated by log rank test. (B) Relative mRNA expression of PFKFB4 in primary tumors and matching metastases in breast cancer patients from GEO: GSE110590 database. p values were calculated by one-way ANOVA and paired t test, two tailed. (C) Immunoblot analyses of PFKFB4 protein expression in LM3.3 human breast cancer cells stably expressing doxycycline inducible non-targeting shRNA (dox-shNT) or two shRNAs targeting PFKFB4 (dox-shPFKFB4 #97 and #99) with or without doxycycline treatment (2 μg/mL). β-actin was used as a loading control. (D) Schematic overview depicting LM3.3 spontaneous metastatic breast cancer model with doxycycline treatment schedule (n = 10). (E) The weight of resected primary tumors (n = 10). Error bars are presented as mean ± SD. ns, not significant, calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (F) Immunoblot analyses of PFKFB4 expression in surgically resected LM3.3 primary tumors after 7 days of doxycycline treatment. Three animal bearing tumors (n = 3) from each group were used for analysis. (G) Representative IVIS image of systemic metastases from each group at day 26 after primary tumor implantation. (H) The kinetics of systemic metastases development after primary tumors were resected, as measured by IVIS imaging (n = 10). Error bars are presented as mean ± SEM. ***p < 0.001 by two-way ANOVA with Dunnett’s multiple comparisons test. See also .

    Journal: Cell reports

    Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

    doi: 10.1016/j.celrep.2022.111756

    Figure Lengend Snippet: (A) Kaplan-Meier survival plots of TNBC breast cancer patients with low or high PFKFB4 signature score in TCGA (n = 149) and GEO: GSE21653 (n = 85) database. Medium cut point of PFKFB4 signature score was used to stratified patients. p values were calculated by log rank test. (B) Relative mRNA expression of PFKFB4 in primary tumors and matching metastases in breast cancer patients from GEO: GSE110590 database. p values were calculated by one-way ANOVA and paired t test, two tailed. (C) Immunoblot analyses of PFKFB4 protein expression in LM3.3 human breast cancer cells stably expressing doxycycline inducible non-targeting shRNA (dox-shNT) or two shRNAs targeting PFKFB4 (dox-shPFKFB4 #97 and #99) with or without doxycycline treatment (2 μg/mL). β-actin was used as a loading control. (D) Schematic overview depicting LM3.3 spontaneous metastatic breast cancer model with doxycycline treatment schedule (n = 10). (E) The weight of resected primary tumors (n = 10). Error bars are presented as mean ± SD. ns, not significant, calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (F) Immunoblot analyses of PFKFB4 expression in surgically resected LM3.3 primary tumors after 7 days of doxycycline treatment. Three animal bearing tumors (n = 3) from each group were used for analysis. (G) Representative IVIS image of systemic metastases from each group at day 26 after primary tumor implantation. (H) The kinetics of systemic metastases development after primary tumors were resected, as measured by IVIS imaging (n = 10). Error bars are presented as mean ± SEM. ***p < 0.001 by two-way ANOVA with Dunnett’s multiple comparisons test. See also .

    Article Snippet: PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining.

    Techniques: Expressing, Two Tailed Test, Western Blot, Stable Transfection, shRNA, Tumor Implantation, Imaging

    (A) Gene sets enriched in WT or PFKFB4-KO EMT6 cells identified in RNA-seq data. Top five gene sets significantly enriched in WT cells are listed. Plot showing gene set enrichment analysis (GSEA) of αvβ3 integrin pathway in EMT6 cells. (B) Heatmap showing differentially expressed genes (DEGs) (p < 0.05, Log 2 FC < −1.5 or > 1.5) in αvβ3 integrin pathway. (C) The mRNA expression of ITGB1, ITGB3 (encoding integrin β3), ITGB5, and ITGAV in EMT6 WT and PFKFB4-KO cells measured by quantitative PCR (n = 3). Error bars are presented as mean ± SD. ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. (D) The protein expression of integrin β3 and PFKFB4 in EMT6 WT and PFKFB4-KO cells measured by immunoblotting. β-actin was used as a loading control. (E) Boyden chamber migration assay using EMT6 WT and PFKFB4-KO cells with serum as chemoattractant (n = 3). Scale bar, 300 μm. Error bars are presented as mean ± SD. ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. (F) Boyden chamber migration assay using EMT6 WT and PFKFB4-KO cells using fibronectin (FN; 10 μg/mL) and vitronectin (VN; 5 μg/mL) as chemoattractant. Scale bar, 300 μm. (G) Quantification of FN- and VN-dependent migration (n = 3). Error bars are presented as mean ± SD. **p < 0.01 by unpaired two-tailed t test. (H) Representative immunohistochemistry images showing integrin β3 (red) expression in shNT and shPFKFB4 metastatic lymph nodes from LM3.3 mouse experiments described in . Human HLA (green) was used as a marker for human tumor cells. Scale bar, 50 μm. (I) Quantification of integrin β3 expression in human HLA-positive cells (n = 3). Error bars are presented as mean ± SD. *p < 0.05 by unpaired two-tailed t test. See also and .

    Journal: Cell reports

    Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

    doi: 10.1016/j.celrep.2022.111756

    Figure Lengend Snippet: (A) Gene sets enriched in WT or PFKFB4-KO EMT6 cells identified in RNA-seq data. Top five gene sets significantly enriched in WT cells are listed. Plot showing gene set enrichment analysis (GSEA) of αvβ3 integrin pathway in EMT6 cells. (B) Heatmap showing differentially expressed genes (DEGs) (p < 0.05, Log 2 FC < −1.5 or > 1.5) in αvβ3 integrin pathway. (C) The mRNA expression of ITGB1, ITGB3 (encoding integrin β3), ITGB5, and ITGAV in EMT6 WT and PFKFB4-KO cells measured by quantitative PCR (n = 3). Error bars are presented as mean ± SD. ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. (D) The protein expression of integrin β3 and PFKFB4 in EMT6 WT and PFKFB4-KO cells measured by immunoblotting. β-actin was used as a loading control. (E) Boyden chamber migration assay using EMT6 WT and PFKFB4-KO cells with serum as chemoattractant (n = 3). Scale bar, 300 μm. Error bars are presented as mean ± SD. ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. (F) Boyden chamber migration assay using EMT6 WT and PFKFB4-KO cells using fibronectin (FN; 10 μg/mL) and vitronectin (VN; 5 μg/mL) as chemoattractant. Scale bar, 300 μm. (G) Quantification of FN- and VN-dependent migration (n = 3). Error bars are presented as mean ± SD. **p < 0.01 by unpaired two-tailed t test. (H) Representative immunohistochemistry images showing integrin β3 (red) expression in shNT and shPFKFB4 metastatic lymph nodes from LM3.3 mouse experiments described in . Human HLA (green) was used as a marker for human tumor cells. Scale bar, 50 μm. (I) Quantification of integrin β3 expression in human HLA-positive cells (n = 3). Error bars are presented as mean ± SD. *p < 0.05 by unpaired two-tailed t test. See also and .

    Article Snippet: PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining.

    Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Migration, Two Tailed Test, Immunohistochemistry, Marker

    (A) Quantitative PCR showing the relative levels of PFKFB4 in MDA-MB-468, MDA-MB-231, and EMT6 cells cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. **p < 0.01; ***p < 0.001 by unpaired two-tailed t test. (B) Wound healing assay using EMT6 WT and PFKFB4-KO cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (C) The mRNA expression of ITGB3 in EMT6 WT and PFKFB4-KO cells under normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (D and E) Immunoblot analyses of integrin β3, HIF-1α, PFKFB4, and β-actin in WT and PFKFB4-KO MDA-MB-468 (D) and EMT6 cells (E) cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ). (F) Immunoblot analyses showing the levels of endogenous integrin β3 and GFP-integrin β3 along with PFKFB4. β-actin was used as a loading control. (G) Boyden chamber migration assay using EMT6 WT, PFKFB4-KO, and PFKFB4-KO cells with ectopic GFP-integrin β3 cultured in hypoxic condition (1% O 2 ). Scale bar, 300 μm. (H) Quantification of migration assay under hypoxic condition (n = 3). Error bars are presented as mean ± SD. ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test. See also .

    Journal: Cell reports

    Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

    doi: 10.1016/j.celrep.2022.111756

    Figure Lengend Snippet: (A) Quantitative PCR showing the relative levels of PFKFB4 in MDA-MB-468, MDA-MB-231, and EMT6 cells cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. **p < 0.01; ***p < 0.001 by unpaired two-tailed t test. (B) Wound healing assay using EMT6 WT and PFKFB4-KO cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (C) The mRNA expression of ITGB3 in EMT6 WT and PFKFB4-KO cells under normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (D and E) Immunoblot analyses of integrin β3, HIF-1α, PFKFB4, and β-actin in WT and PFKFB4-KO MDA-MB-468 (D) and EMT6 cells (E) cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ). (F) Immunoblot analyses showing the levels of endogenous integrin β3 and GFP-integrin β3 along with PFKFB4. β-actin was used as a loading control. (G) Boyden chamber migration assay using EMT6 WT, PFKFB4-KO, and PFKFB4-KO cells with ectopic GFP-integrin β3 cultured in hypoxic condition (1% O 2 ). Scale bar, 300 μm. (H) Quantification of migration assay under hypoxic condition (n = 3). Error bars are presented as mean ± SD. ***p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test. See also .

    Article Snippet: PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining.

    Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Two Tailed Test, Wound Healing Assay, Expressing, Western Blot, Migration

    (A) Representative images of ultrasound and photoacoustic imaging (US-PAI) from LM3.3 shNT and shPFKFB4 primary tumors. The images were acquired in 3D-mode and presented in three representative frames showing the oxygen saturation (sO 2 %) from the periphery (left and right frames) and the core (middle frame) of the tumors. (B) Quantification of oxygen saturation (tumor 3D sO 2 %) in shNT and shPFKFB4 tumors (n = 3). Error bars are presented as mean ± SD. ns, not significant, calculated by unpaired two-tailed t test. (C) Immunohistochemical staining of PFKFB4 (red) and pimonidazole (green) from resected LM3.3 shNT and shPFKFB4 primary tumors (n = 4). Pimonidazole isa marker of hypoxia. Each tumor was imaged and quantified in both pimonidazole negative and positive areas. Scale bar, 75 μm. (D) Quantification of the intensity of PFKFB4 staining (n = 4). Error bars are presented as mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (E) Experimental workflow of in vitro (n = 3) and in vivo (n = 5) targeted metabolomics. (F) Principal component analysis of targeted metabolomics from EMT6 WT and PFKFB4-KO cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ). (G) Relative levels of serine, glycine, aspartate, arginine, and GSH/GSSG in WT and PFKFB4-KO EMT6 cells cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (H) Unique metabolic signature regulated by PFKFB4 in TNBC is schematically demonstrated. See also and .

    Journal: Cell reports

    Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

    doi: 10.1016/j.celrep.2022.111756

    Figure Lengend Snippet: (A) Representative images of ultrasound and photoacoustic imaging (US-PAI) from LM3.3 shNT and shPFKFB4 primary tumors. The images were acquired in 3D-mode and presented in three representative frames showing the oxygen saturation (sO 2 %) from the periphery (left and right frames) and the core (middle frame) of the tumors. (B) Quantification of oxygen saturation (tumor 3D sO 2 %) in shNT and shPFKFB4 tumors (n = 3). Error bars are presented as mean ± SD. ns, not significant, calculated by unpaired two-tailed t test. (C) Immunohistochemical staining of PFKFB4 (red) and pimonidazole (green) from resected LM3.3 shNT and shPFKFB4 primary tumors (n = 4). Pimonidazole isa marker of hypoxia. Each tumor was imaged and quantified in both pimonidazole negative and positive areas. Scale bar, 75 μm. (D) Quantification of the intensity of PFKFB4 staining (n = 4). Error bars are presented as mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (E) Experimental workflow of in vitro (n = 3) and in vivo (n = 5) targeted metabolomics. (F) Principal component analysis of targeted metabolomics from EMT6 WT and PFKFB4-KO cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ). (G) Relative levels of serine, glycine, aspartate, arginine, and GSH/GSSG in WT and PFKFB4-KO EMT6 cells cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ) (n = 3). Error bars are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (H) Unique metabolic signature regulated by PFKFB4 in TNBC is schematically demonstrated. See also and .

    Article Snippet: PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining.

    Techniques: Imaging, Two Tailed Test, Immunohistochemical staining, Staining, Marker, In Vitro, In Vivo, Cell Culture

    (A and B) Representative PFKFB4 immunohistochemical staining of breast cancer patients (n = 122) with high or low staining of PFKFB4 in the nucleus (A) and cytoplasm (B). Scale bar, 300 μm. (C and D) Kaplan-Meier analysis of the overall survival of breast cancer patients (n = 122) with high or low expression of PFKFB4 in the nucleus (C) or cytoplasm (D). (E) WT and PFKFB4-KO MDA-MB-468 cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) were subjected to subcellular fractionation, followed by immunoblot analyses of PFKFB4, β-tubulin, and lamin A/C. PFKFB4 blot was imaged for 30 s to obtain a low exposure and 120 s to obtain a high exposure. (F) Immunoblot analyses of nuclear fractions from MDA-MB-468 WT and PFKFB4-KO cells cultured in normoxic or hypoxic conditions were probed with PFKFB4, lamin A/C, and HIF-1α antibodies. (G) MDA-MB-468 cells cultured in normoxic or hypoxic conditions were used for immunofluorescence staining of PFKFB4. Scale bar, 25 μm (low-magnification image) and 10 μm (high-magnification image). (H and I) The mRNA expression of SLC2A1 (H) and PDK1 (I) in WT and PFKFB4-KO MDA-MB-468 cells under normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) was measured by quantitative PCR (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (J) HIF-1α luciferase (HRE) reporter assay in MDA-MB-468 WT and PFKFB4-KO cells expressing shNT or shSRC-3 cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) for 24 h. Error bars are presented as mean ± SD. ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (K) Immunoblot analyses of SRC-3 and integrin β3 in MDA-MB-231 cells stably expressing shNT or shSRC-3. β-actin was used as a loading control. See also and and .

    Journal: Cell reports

    Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

    doi: 10.1016/j.celrep.2022.111756

    Figure Lengend Snippet: (A and B) Representative PFKFB4 immunohistochemical staining of breast cancer patients (n = 122) with high or low staining of PFKFB4 in the nucleus (A) and cytoplasm (B). Scale bar, 300 μm. (C and D) Kaplan-Meier analysis of the overall survival of breast cancer patients (n = 122) with high or low expression of PFKFB4 in the nucleus (C) or cytoplasm (D). (E) WT and PFKFB4-KO MDA-MB-468 cells cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) were subjected to subcellular fractionation, followed by immunoblot analyses of PFKFB4, β-tubulin, and lamin A/C. PFKFB4 blot was imaged for 30 s to obtain a low exposure and 120 s to obtain a high exposure. (F) Immunoblot analyses of nuclear fractions from MDA-MB-468 WT and PFKFB4-KO cells cultured in normoxic or hypoxic conditions were probed with PFKFB4, lamin A/C, and HIF-1α antibodies. (G) MDA-MB-468 cells cultured in normoxic or hypoxic conditions were used for immunofluorescence staining of PFKFB4. Scale bar, 25 μm (low-magnification image) and 10 μm (high-magnification image). (H and I) The mRNA expression of SLC2A1 (H) and PDK1 (I) in WT and PFKFB4-KO MDA-MB-468 cells under normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) was measured by quantitative PCR (n = 3). Error bars are presented as mean ± SD. *p < 0.05; ***p < 0.001; ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. (J) HIF-1α luciferase (HRE) reporter assay in MDA-MB-468 WT and PFKFB4-KO cells expressing shNT or shSRC-3 cultured in normoxic (21% O 2 ) or hypoxic condition (1% O 2 ) for 24 h. Error bars are presented as mean ± SD. ****p < 0.0001; ns, not significant, calculated by one-way ANOVA with Sidak’s multiple comparisons test. (K) Immunoblot analyses of SRC-3 and integrin β3 in MDA-MB-231 cells stably expressing shNT or shSRC-3. β-actin was used as a loading control. See also and and .

    Article Snippet: PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining.

    Techniques: Immunohistochemical staining, Staining, Expressing, Cell Culture, Fractionation, Western Blot, Immunofluorescence, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Stable Transfection

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

    doi: 10.1016/j.celrep.2022.111756

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: PFKFB4 Antibody (Abcam, Cat. No. ab137785) at 1/100 was used for staining.

    Techniques: Cell Culture, Produced, Recombinant, Infection, Western Blot, Lysis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Blocking Assay, Stripping, Magnetic Beads, SYBR Green Assay, Luciferase, Amplification, Sequencing, shRNA, Software, Real-time Polymerase Chain Reaction