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

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: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Confocal Microscopy

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

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

Journal: Cancers

Article Title: Characterization of Estrogen Receptors in Pancreatic Adenocarcinoma with Tertiary Lymphoid Structures

doi: 10.3390/cancers15030828

Figure Lengend Snippet: Correlation between ERs expression and tumor-infiltrating immune cells in PAAD. ( A ) Representative IHC images of ERα (+) PAAD tumor tissues with concurrent intratumoral TLS structures; TLS was circled in red line; scale bar: 20 µm. ( B ) Pie plots of the proportion of TLS (+) samples in ERα (+) and ERα (−) groups. ( C ) Histograms of relative infiltration levels of CD4+T-cells, CD8 + T-cells, HLA-DR+ activated immune cells, and FOXP3+ Tregs in ERα (+) and ERα (−) groups; Treg: regulatory T-cell; ** p < 0.01, *** p < 0.001, ns: non-significant. ( D ) Representative IHC images of ERβ (+) PAAD tumor tissues with concurrent intratumoral TLS structures; TLS was circled in red line; scale bar: 20 µm. ( E ) Pie plots of the proportion of TLS (+) samples in ERβ (+) and ERβ (−) groups. ( F ) Histograms of relative infiltration levels of CD4+T-cells, CD8 + T-cells, HLA-DR+ activated immune cells, and FOXP3+ Tregs in ERα (+) and ERα (−) groups; * p < 0.05, ** p < 0.01, ns: non-significant. ( G ) Representative IHC images of GPER (+) PAAD tumor tissues with concurrent intratumoral TLS structures; TLS was circled in red line; scale bar: 20 µm. ( H ) Pie plots of the proportion of TLS (+) samples in GPER (+) and GPER (−) groups. ( I ) Histograms comparing relative infiltration levels of CD4+T-cells, CD8 + T-cells, HLA-DR+ activated immune cells and FOXP3+ Tregs in ERα (+) and ERα (−) groups; ns: non-significant.

Article Snippet: The primary antibodies used in this study included anti-ERα antibody (1/200, pH 6.0, No. ab79413, Abcam, Waltham, MA, USA), anti-ERβ antibody (1/200, pH 9.0, No. ab288, Abcam, Waltham, MA, USA), anti-GPER antibody (1/200, pH 9.0, No. ab260033, Abcam, Waltham, MA, USA), anti-CD4 antibody (1/200, pH 9.0, No. ab133616, Abcam, Waltham, MA, USA), anti-CD8 antibody (1/100, pH 9.0, No. ab178089, Abcam, Waltham, MA, USA), anti-CD20 antibody (1/100, pH 6.0, No. ab64088, Abcam, Waltham, MA, USA), anti-HLA-DR-antibody (1/200, pH 6.0, No. ab20181, Abcam, Waltham, MA, USA), and anti-FOXP3 antibody (1/100, pH 9.0, No. ab20034, Abcam, Waltham, MA, USA).

Techniques: Expressing

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

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

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

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

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

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