Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Expression of HLA-G in tumor and normal tissues and cells. (A) Expression of mRNA encoding HLA-G in matched tumor and normal tissues was extracted from the GEPIA databases and presented as boxplots. (B) HLA-G protein expression in tumor lesions (upper panel) and matched healthy tissues (lower panel) from patients with TNBC, GBM, PA, and OV cancers, as determined by immunohistochemical analysis with anti-HLA-G antibodies. The primary human hepatocyte formalin-fixed, paraffin-embedded (FFPE) cell pellets with or without HLA-G-expressing plasmid transfection as staining controls (middle panel). (C) Representative micrographs of HLA-G proteins on a normal tissue microarray. Tissue sections were stained with anti-HLA-G antibodies. Scale bars, 50 µm. Original magnification: ×400. The expression of HLA-G on cell membrane was counted and presented as H score. (D) Cell surface expression of HLA-G by solid tumor cell lines and immortalized non-malignant cell lines. MDA-MB-231, DBTRG-05MG, AsPC-1, SKOV3, HUVEC, SVGp12, and BEAS-2b cells and primary hepatocytes, were stained with FITC-conjugated anti-HLA-G antibodies or with isotype-matched control antibodies, and flow cytometry analysis was conducted using the FL1 channel. The results are presented as histograms. GBM, glioblastoma; HLA-G, human leukocyte antigen G; HUVEC, human umbilical vein endothelial cell; OV, ovarian cancer; PA, pancreatic cancer; TNBC, triple-negative breast cancer.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: Expressing, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Plasmid Preparation, Transfection, Staining, Microarray, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Cytotoxic killing of multiple solid tumor cell lines by anti-HLA-G CAR-NK cells. (A) Lentiviral vector map of the DAP12-based anti-HLA-G CAR construct. Schematic representation of the anti-HLA-G antibody scFv fragment linked with the transmembrane and intracellular domain of KIR2DS4 following self-cleavage peptide P2A and full-length DAP-12, fused to the suicide iC9 protein. (B) Purity, transduction efficiency and phenotypical analysis of anti-HLA-G CAR-NK cells. NK cells were transduced (or not) with anti-HLA-G CAR lentiviral particles, and expression of anti-HLA-G CAR was determined by protein L staining (upper left panel). Expression of LILRB1 and KIR2DL4 in CD56-positive cells (upper right panel), the populations of CD56, CD16, CD3, CD19, CD11b, and CD66b in CD45-positive cells (middle left panel), the frequency of CD94, NKG2D, Nkp44, Nkp46, and Nkp30 in CD56-positive cells (middle right panel), and the expression of CD56 and CD16 were analyzed by flow cytometry using specific fluorescent-conjugated antibodies (bottom left panel). The secretion of granzyme B, perforin, IFN-γ, and TNF-α form NK cells with or without MDA-MB-231 cells challenge (E:T as 1:1) were measured by ELISA assays. (C) Schematic showing the gating strategy used to determine tumor cell death (PI/annexin V staining) triggered by anti-HLA-G CAR-NK cells. (D) MDA-MB-231, DBTRG-05MG, AsPC-1, and SKOV3 cells were incubated with parental or anti-HLA-G CAR-transduced NK cells for 24, 48, and 72 hours at E:T ratios of 0.1:1.0, 1:1, 1:3, 1:6, and 1:10. (E) Characterization of stable HLA-G knockdown AsPC-1 and MDA-MB-231 cell lines by flow cytometry (upper right) and immunoblotting (lower right) with specific antibodies; the corresponding HLA-G CAR-NK-induced cytotoxicity for 48 hours at E:T ratios of 1:1, 1:3, and 1:6 was determined by flow cytometry using PI/annexin V staining (left). Tumor cell death was determined by flow cytometry after PI/annexin V staining. Data are expressed as the mean±SEM of ≥3 independent experiments (*p<0.05, **p<0.01, ***p<0.001). CAR, chimeric antigen receptor; E:T, effector:target; HLA-G, human leukocyte antigen G; IFN-γ, interferon gamma; NK, natural killer; scFv, single-chain variable fragment; TNF-α, tumor necrosis factor alpha.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: Plasmid Preparation, Construct, Transduction, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Anti-HLA-G CAR construct switches tumorous HLA-G signaling in NK cells from inhibitory to activating. (A) Overexpression of HLA-G in MDA-MB-231 cells. HLA-G levels in control and HLA-G-expressing vector-transfected MDA-MB-231 cells were determined by western blot analysis and flow cytometry using specific antibodies (see the histogram overlays). (B–D) Control and HLA-G-overexpressing MDA-MB-231 cells were cocultured with anti-HLA-G CAR-NK or mock-transfected NK cells at an E:T ratio of 1:1. After 48 hours of coculture, the cell-killing rate (B), the expression of FasL, TRAIL, and CD107a (C), and the secretion of granzyme B, perforin, IFN-γ, and TNF-α (D) of NK cells was measured by flow cytometry after PI/annexin V staining and staining with specific antibodies, respectively. (E) NK cells were harvested after 1 hour of coculture with parental or HLA-G-overexpressing MDA-MB-231 cells, and the expression of phosphorylated SHP-1 and Syk/ZAP70 were determined by intracellular staining using specific antibodies then analyzed by flow cytometry. (F) Molecular interactions between the HLA-G on tumor cells, endogenous HLA-G receptors LILRB1, and KIR2DL4, and the anti-HLA-G CAR construct on engineered NK cells. Data are expressed as the mean±SEM of ≥3 independent experiments (*p<0.05, **p<0.01, ***p<0.001). CAR, chimeric antigen receptor; Ctrl, control; E:T, effector:target; HLA-G, human leukocyte antigen G; IFN-γ, interferon gamma; NK, natural killer; TNF-α, tumor necrosis factor alpha.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: Construct, Over Expression, Expressing, Plasmid Preparation, Transfection, Western Blot, Flow Cytometry, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Chemotherapeutic agents upregulate membrane expression of HLA-G on tumor cell lines but not normal or normal-transformed cell lines. (A–C) Tumor cell surface expression of HLA-G after treatment with chemotherapeutic agents. MDA-MB-231, DBTRG-05MG, AsPC-1, and SKOV3 cells were treated for 48 hours with 200 nM Dox, 80 µg/mL TMZ, 20 µg/mL Gem, or 20 µM CBP, respectively. Cells were collected and analyzed to assess subcellular localisation of HLA-G and pan-cadherin using immunocytochemistry and confocal microscopy (A); scale bars, 37 µm. Cell surface expression of HLA-G is shown in the histogram overlays (B). Expression of HLA-G, E-cadherin, and β-actin in the cytosol and membrane fractions, as assessed by immunoblotting with specific antibodies. The primary human hepatocytes transfected with or without HLA-G expression plasmid as staining controls (C). (D) Expression of HLA-G expression on normal or normal-transformed cell lines following chemotherapy. HUVECs, primary hepatocytes, BEAS-2b cells, and SVGp12 cells were treated for 48 hours with or without 200 nM Dox, 80 µg/mL TMZ, 20 µg/mL Gem, and 20 µM CBP, respectively. Cells were harvested and HLA-G expression was analyzed using flow cytometry after staining with specific antibodies (see the histogram overlays). Data are expressed as the mean±SEM of ≥3 independent experiments (*p<0.05, **p<0.01, ***p<0.001). HLA-G, human leukocyte antigen G; HUVEC, human umbilical vein endothelial cell.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: Expressing, Transformation Assay, Immunocytochemistry, Confocal Microscopy, Western Blot, Transfection, Plasmid Preparation, Staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Genetic/epigenetic regulation of TAP-1 and genetic expression of SPP in tumor cells treated with chemotherapeutic agents affect cell surface expression of HLA-G via altered Golgi trafficking. (A) MDA-MB-231 and SKOV3 cells were treated (or not) for 24 hours with 200 nM Dox or 20 µM CBP, followed by incubation with 2 µg/mL BFA for an additional 16 hours. Cell surface expression of HLA-G was determined by flow cytometry. (B) MDA-MB-231 or DBTRG-05MG cells were transfected for 48 hours with control, TAP-1, or SPP siRNA, and then treated with 200 nM Dox or 80 µg/mL TMZ. Expression of HLA-G, TAP-1, SPP, and β-actin in the membrane and cytosolic fractions was determined by immunoblotting (upper panels). Relative cell surface expression of HLA-G was evaluated by flow cytometry (lower panel). (C) MDA-MB-231, DBTRG-05MG, AsPC-1, and SKOV3 cells were treated (or not) with 200 nM Dox, 80 µg/mL TMZ, 20 µg/mL Gem, or 20 µM CBP for 48 hours, and TAP-1 gene promoter methylation status was analyzed by methylated-MSP and unmethylated-MSP PCR. (D) DNMT1 expression in chemotherapeutic agent-treated tumor cells. MDA-MB-231, DBTRG-05MG, AsPC-1, and SKOV3 cells were treated (or not) with 200 nM Dox, 80 µg/mL TMZ, 20 µg/mL Gem, or 20 µM CBP for 48 hours. Expression of DNMT-1 and β-actin was analyzed by immunoblotting (upper panel); relative expression (measured by western blotting) is shown in the lower panels. (E, F) Control and DNMT1-overexpressing MDA-MB-231 cells were treated (or not) with Dox (200 nM) for 48 hours, and expression of DNMT1 in control and DNMT1-overexpressing MDA-MB-231 cells was detected by immunoblotting (E). The methylation status of the TAP-1 gene promoter was analyzed by methylated-MSP and unmethylated-MSP PCR (F). (G) Control and DNMT1-overexpressing MDA-MB-231 cells were treated (or not) with Dox (200 nM) for 48 hours, and cell surface expression of HLA-G was determined by flow cytometry after staining with specific antibodies. (H, I) Expression of DNMT1 affects sensitivity to anti-HLA-G CAR-NK. Control and DNMT-overexpressing MDA-MB-231 cells were pretreated (or not) with Dox (50 nM) for 48 hours and then incubated with anti-HLA-G CAR-NK or mock NK cells for an additional 48 hours. Cell killing by NK cells was measured by flow cytometry after PI/annexin V staining (H), the secreted granzyme B, perforin, IFN-γ, and TNF-α were detected by ELISA (I). (J) Schematic representation for the mechanism by which chemotherapy-induced increases HLA-G expression and cytokines secreted by CAR-NK cells. Data are expressed as the mean±SEM of at least three independent experiments (*p<0.05, **p<0.01, ***p<0.001). BFA, brefeldin A; CAR, chimeric antigen receptor; CBP, carboplatin; Ctrl, control; Dox, doxorubicin; Gem, gemcitabine; HLA-G, human leukocyte antigen G; IFN-γ, interferon gamma; MSP, methylation-specific PCR; NK, natural killer; SPP, signal peptide peptidase; TMZ, temozolomide; TNF-α, tumor necrosis factor alpha.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: Expressing, Incubation, Flow Cytometry, Transfection, Western Blot, Methylation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Pretreatment with chemotherapeutic agents sensitizes tumor cells but not non-malignant cells, to anti-HLA-G CAR-NK-induced cell death. (A) MDA-MB-231, DBTRG-05MG, AsPC-1, and SKOV3 cells were pretreated with 50 nM Dox, 40 µg/mL TMZ, 10 µg/mL Gem, and 10 µM CBP, respectively, for 48 hours. Subsequently, cells were incubated with mock control or HLA-G CAR-NK cells for 24, 48, or 72 hours. Tumor cell death was assessed at the indicated times by flow cytometry after PI/annexin V staining. (B) FasL, TRAIL, and CD107a expression on NK cells was detected by flow cytometry after staining with specific antibodies. (C) Granzyme B, perforin, IFN-γ, and TNF-α contents in supernatants collected from NK and tumor cell cocultures were measured by ELISA. (D, E) Cytotoxicity effects of HLA-G CAR-NK or mock NK cells against stable HLA-G knockdown MDA-MB-231 cells were determined after 48 hours of coculture by flow cytometry after staining with PI/annexin V staining (D), and the contents of granzyme B, perforin, IFN-γ, and TNF-α in supernatants were analyzed by ELISA. (F) HUVECs, primary bone marrow cells, primary hepatocytes, BEAS-2b cells, and SVGp12 cells were pretreated for 48 hours with the same dose of chemotherapeutic agents used for the aforementioned tumor cell lines, after which they were incubated for 24, 48, or 72 hours with parental or HLA-G CAR-NK cells from donors. Cell death was evaluated by flow cytometry after PI/annexin V staining. Data are expressed as the mean±SEM of ≥3 independent experiments (*p<0.05, **p<0.01, ***p<0.001). CAR, chimeric antigen receptor; CBP, carboplatin; Dox, doxorubicin; Gem, gemcitabine; HLA-G, human leukocyte antigen G; HUVEC, human umbilical vein endothelial cell; IFN-γ, interferon gamma; NK, natural killer; TMZ, temozolomide; TNF-α, tumor necrosis factor alpha.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: Incubation, Flow Cytometry, Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

doi: 10.1136/jitc-2021-003050

Figure Lengend Snippet: Pretreatment with Dox and TMZ enhances the in vivo antitumor activity of anti-HLA-G CAR-NK cells. (A) Treatment protocol used for the TNBC animal model. MDA-MB-231 cells (1×10 6 /mouse) were implanted orthotopically on day 0. On day 6 and for the following 3 weeks, mice received a weekly injection of normal saline or 0.5 mg/kg Dox via the tail vein. On day 7, mice were injected with normal saline or with 1.5×10 7 mock or anti-HLA-G CAR-NK cells, followed by infusion of 5×10 6 mock or anti-HLA-G CAR-NK cells each subsequent week for 3 weeks. (B) Representative IVIS images of MDA-MB231 tumors treated according to the protocols described in (A). Images were taken weekly. (C) Tumor progression, as measured by bioluminescence photometry. The luminoscore was calculated as the sum of flux values from the front and back views. (D) Survival was plotted using the Kaplan-Meier method. Mice were considered dead when the tumor volume exceeded 2000 mm 3 or the greatest dimension was >1.5 cm in any direction, as measured using an electronic manual caliper. (E) Treatment protocol for the GBM animal model. U87 cells (1×10 6 /mouse) were implanted intracranially on day 0. On day 6 and for the following 3 weeks, mice were treated weekly (or not) with 3 mg/kg TMZ by oral gavage. Mice were injected with normal saline, or 1.5×10 7 mock or anti-HLA-G CAR-NK cells, on day 7, followed by infusion of 5×10 6 mock or anti-HLA-G CAR-NK cells each subsequent week for 3 weeks. (F) Representative weekly IVIS images of U87 tumors treated according to the protocols described in (E). (G) Tumor progression, as measured by bioluminescence photometry. (H) Survival plotted using the Kaplan-Meier method (*p<0.05, **p<0.01, ***p<0.001). CAR, chimeric antigen receptor; Dox, doxorubicin; GBM, glioblastoma; HLA-G, human leukocyte antigen G; IL, interleukin; NK, natural killer; TMZ, temozolomide; TNBC, triple-negative breast cancer.

Article Snippet: To generate stable clones, HLA-G small hairpin RNA plasmids (Santa Cruz Biotechnology) and HLA-G and DNMT1 expression plasmids (Origene) were transfected for 48 hours into MDA-MB-231 and AsPC-1 cells using Lipofectamine 3000.

Techniques: In Vivo, Activity Assay, Animal Model, Injection

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: Increased HKDC1 expression is regulated by PGC1β in breast cancer cells. Different cells, including primary HMECs, MCF7 and MDA231 cells, were infected by either empty (CTL) or shPGC1β lentivirus, and the cells were harvested for gene expression analysis. (A) mRNA level by qPCR, n = 4. (B) Quantitation of protein levels, n = 5. (C) Representative pictures for Western Blotting. * , P < 0.05, vs. HMECs/CTL group; ¶, P < 0.05 vs. MCF7/CTL group; #, P < 0.05, vs. MDA231/CTL group. Results are expressed as mean ± SEM.

Article Snippet: MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics.

Techniques: Expressing, Infection, Gene Expression, Quantitation Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: HKDC1 expression is regulated by PGC1β/SREBP1-mediated co-activation on the HKDC1 promoter. (A) The MCF7 cells were infected by either PGC1β (↑PGC1β) or empty control (CTL) lentivirus for 2 days, and then the cells were transiently transfected by either HKDC1 full length (pLDHA-2000) or deletion reporter plasmids. After 24 h, the PGC1β-induced HKDC1 reporter activities from PGC1β lentivirus infected cells (↑PGC1β) were calculated as the relative percentage (% control) by comparing to lentivirus empty control (CTL) infected cells. *, P < 0.05, vs. pHKDC1-2000 group, n = 5. (B) The schematic picture for the potential transcriptional binding motif in the range of−1300~-1200 (from transcription start site) on the HKDC1 promoter, and the potential SREBP1 binding site was marked in red. (C) The lentivirus-infected MCF7 cells were transiently transfected by either HKDC1 full length (pHKDC1-2000) or the specific transcriptional binding motif mutation reporter plasmids, and then the reporter activities were measured after 24 h. *, P < 0.05, vs. pHKDC1-2000 group, n = 5. (D) The lentivirus infected MCF7 cells were transiently transfected by HKDC1 full length (pHKDC1-2000) or truncate (pHKDC1-1200 and pHKDC1-0) reporters, or SREBP1 (located at the site of−1286) deletion plasmid [Δ-1286(SREBP1)], and after 24 h, the reporter activities were measured. *, P < 0.05, vs. pHKDC1-2000 group, n = 5. (E) Different breast cancer cells were used for ChIP analysis by PGC1β, SREBP1, or SREBP2 antibody, respectively, and the HKDC1 promoter in the range of−1300~-1100 was amplified and measured by qPCR, n = 5. *, P < 0.05, vs. HMECs/CTL group; ¶, P < 0.05 vs. MCF7/CTL group; #, P < 0.05, vs. MDA231/CTL group. (F) The MCF7 cells were transfected by siRNA for either non-sense control (CTL), PGC1β, SREBP1, or SREBP2 for 2 days, and the cells were harvested for mRNA analysis. *, P < 0.05, vs. CTL group, n = 5. Results are expressed as mean ± SEM.

Article Snippet: MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics.

Techniques: Expressing, Activation Assay, Infection, Control, Transfection, Binding Assay, Mutagenesis, Plasmid Preparation, Amplification

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: HKDC1 expression modulates oxidative stress, apoptosis and mitochondrial function in MCF7 cells. The MCF7 cells were infected by either expression or knockdown lentivirus for either PGC1β or HKDC1, and the subsequent stable cell lines or related empty vector control (CTL) were cultured for 2 days, and then the cells were harvested for further analysis. (A) Representative pictures for the morphological changes of treated MCF7 cells. (B) mRNA level by qPCR, n = 4. (C) Quantitation of protein levels, n = 5. (D) Representative pictures for Western Blotting. (E) ROS formation, n = 5. (F) 3-Nitrotyrosine formation, n = 5. (G) Apoptosis rate by TUNEL assay, n = 5. (H) Caspase-3 activity, n = 5. (I) Mitochondrial DNA copies, n = 4. (J) Intracellular ATP level, n = 5. *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. Results are expressed as mean ± SEM.

Article Snippet: MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics.

Techniques: Expressing, Infection, Knockdown, Stable Transfection, Plasmid Preparation, Control, Cell Culture, Quantitation Assay, Western Blot, TUNEL Assay, Activity Assay

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: HKDC1 expression modulates permeability transition pore and glucose uptake by binding with VDAC1 in MCF7 cells. The MCF7 cells were infected by either expression or knockdown lentivirus for either PGC1β or HKDC1, and the subsequent stable cell lines or related empty vector control (CTL) were cultured for 2 days, then the cells were harvested for further analysis. (A) Mitochondrial membrane potential (ΔΨm), n = 5. (B) [ 3 H]-deoxyglucose uptake, n = 4. (C) The treated cells were IP by VDAC1, then IB by HKDC1, and VDAC1, with 10% of β-actin from whole lysates as input control. (D) Protein quantitation for (C) , n = 4. (E) The MCF7 cells were infected by either lentivirus empty control (CTL) or shHKDC1, then the cells were immunostained by HKDC1 (green), and the mitochondria was stained by MitoTracker Red (red). *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. Results are expressed as mean ± SEM.

Article Snippet: MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics.

Techniques: Expressing, Permeability, Binding Assay, Infection, Knockdown, Stable Transfection, Plasmid Preparation, Control, Cell Culture, Membrane, Protein Quantitation, Staining

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: HKDC1 expression potentiates cell proliferation, while HKDC1 knockdown reverses this effect in MCF7 cells. The MCF7 cells were infected by either expression or knockdown lentivirus for either PGC1β or HKDC1, and the subsequent stable cell lines or related empty vector control (CTL) were cultured for 2 days, and then the cells were harvested for further analysis. (A) Cell proliferation analysis by thymidine incorporation, n = 5. (B) Colony formation assay in soft agar, n = 5. (C) Representative pictures for (B) . (D) Quantitation of Ki-67 positive cells, n = 3. (E) Cell invasion and migration assay, n = 4. (F) Representative picture for (E) . *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. Results are expressed as mean ± SEM.

Article Snippet: MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics.

Techniques: Expressing, Knockdown, Infection, Stable Transfection, Plasmid Preparation, Control, Cell Culture, Colony Assay, Quantitation Assay, Migration

Journal: Frontiers in Oncology

Article Title: PGC1β Regulates Breast Tumor Growth and Metastasis by SREBP1-Mediated HKDC1 Expression

doi: 10.3389/fonc.2019.00290

Figure Lengend Snippet: HKDC1 expression involves with tumor growth and metastasis in vivo . (A,B) Tissue Microarray slides (#CHTN BrCaProg1) were obtained from CHTN (Cooperative Human Tissue Network), and the immunohistochemistry was performed using a primary rabbit antibody for either PGC1β or HKDC1 and a second anti-rabbit- FITC. Sixty cells from normal breast tissue (normal), primary tumor (tumor), and metastatic (metastasis) tissues were quantitated by Image J. (A) Protein quantitation, n = 3. *, P < 0.05, vs. Normal group; ¶, P < 0.05 vs. Tumor group. (B) Representative pictures for (A) . (C–H) The nude mice were injected with treated MCF7 cells through the tail vein for in vivo xenograft tumor development study, and the treated mice were sacrificed for further analysis. (C) The tumor tissues from the lung were isolated for mRNA analysis by qPCR, n = 4. *, P < 0.05, vs. CTL group. (D) Superoxide anion release from tumor tissues, n = 5, *, P < 0.05, vs. CTL group; #, P < 0.05, vs. shPGC1β group. (E–G) Mice were killed upon 20% weight loss, and the lungs were harvested for terminal analysis. The metastatic tumor nodules from the lungs were counted, and then the formalin-fixed, paraffin-embedded tumor tissue of the lung was sectioned to 4 mm thickness, and the histopathological analyses were performed with H&E staining. Images were taken using a Carl Zeiss MIRAX MIDI slide scanner, and the lung tumor spots were analyzed using a 3DHISTECH Pannoramic Viewer. (E) Tumor colony formation in lung, n = 9. *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. (F) Quantitated lung tumor spots, n = 5. *, P < 0.05, vs. CTL group; ¶, P < 0.05, vs. ↑PGC1β group; #, P < 0.05, vs. shPGC1β group. (G) Representative picture by H&E staining. (H) Kaplan-Meier analysis comparing survival of mice between each treatment group, P -value represents log-rank Mantel-Cox test result, n = 9. Results are expressed as mean ± SEM.

Article Snippet: MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics.

Techniques: Expressing, In Vivo, Microarray, Immunohistochemistry, Protein Quantitation, Injection, Isolation, Formalin-fixed Paraffin-Embedded, Staining