Journal: Theranostics

Article Title: Histone H4K8 lactylation promotes glioblastoma progression by inducing NUPR1-mediated autophagosome‒lysosome fusion

doi: 10.7150/thno.126579

Figure Lengend Snippet: H4K8la directly binds to the NUPR1 promoter and activates its transcription. (A-C) CUT&Tag analysis using an anti-H4K8la antibody revealed a significant reduction in H4K8la enrichment at transcription start sites (TSSs) and promoter regions in oxamate-treated GBM cells. (D) KEGG pathway analysis of genes whose H4K8la promoter occupancy was reduced in the oxamate-treated group. (E) Venn diagram showing that NUPR1, a key autophagy-related gene, is downregulated by oxamate and bound by H4K8la. (F) Western blot analysis showing changes in NUPR1, LDHA, LDHB, and H4K8la levels in GBM cells after LDHA/LDHB knockdown and NaLa rescue treatment. (G) Integrative Genomics Viewer tracks of CUT&Tag showing enriched H4K8la in the promoters of NUPR1. (H) DNA fragments were immunoprecipitated with the H4K8la antibody and analysed by gel electrophoresis and qPCR. (I) Western blot showing alterations in NUPR1 expression in GBM cells following oxamate treatment and oeNUPR1 transfection. (J) CCK-8 assay revealing changes of GBM#P3 and LN229 cells after oxamate treatment and oeNUPR1 transfection in the cell viability. (K) IHC images and statistical analysis showing NUPR1 expression in gliomas of different grades and NBT. (L) IF staining and statistical graphs showing NUPR1 expression and subcellular localization in NBT and gliomas of different grades. Data are presented as mean ± SD. Significance levels between specified treatment groups are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cell lysates were first incubated overnight at 4 °C with antibodies against NUPR1 (15056-1-AP, Proteintech), P300 (Santa Cruz Biotechnology, SC-48343), HDAC2 (Santa Cruz Biotechnology, SC-9959), VPS33A (Proteintech, 16896-1-AP), H4K8la (PTM-1415, PTM BIO, 1:1000) or control IgG (Rabbit IgG: 30000-0-AP, Proteintech; Mouse IgG, B900620, Proteintech).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Nucleic Acid Electrophoresis, Expressing, Transfection, CCK-8 Assay, Staining

Journal: Theranostics

Article Title: Histone H4K8 lactylation promotes glioblastoma progression by inducing NUPR1-mediated autophagosome‒lysosome fusion

doi: 10.7150/thno.126579

Figure Lengend Snippet: NUPR1 expression is regulated by lactylation and promotes GBM malignancy. (A) The UMAP visualization delineates the heterogeneous cellular composition of glioma tissue. (B)The UMAP plot illustrates the nine identified distinct tumor cell clusters. (C, D) UMAP and bar plot display the expression level of NUPR1 in malignant cell clusters. (E) Malignant cells were divided into Low NUPR1 expression cells (Low-NUPR1) and High NUPR1 expression cells (High-NUPR1). (F) The volcano plot displays the upregulated genes in High-NUPR1 cells and labels the top 20 genes with the highest absolute log2 fold change. (G) KEGG pathway activity scores demonstrate differences in metabolic pathways between glioma cells with high and low NUPR1 expression levels. (H) The effects of NUPR1 knockdown on the proliferation of LN229 cells were determined via an EdU staining assay with images and quantitative analysis (scale bars: 100 μm). (I, J) Representative images and quantitative analysis of the Transwell invasion assays showing the migration and invasive capacity of LN229 cells treated with siNUPR1 (scale bars: 200 μm). (K) 3D invasive spheroid assay results demonstrating the distant invasive capacity of GBM#P3 cells after siNUPR1 treatment (scale bars: 300 μm). (L) Following orthotopic implantation of luciferase-GBM#P3 cells into athymic nude mice, we monitored tumor growth by bioluminescence detection using an IVIS-200 imaging system. (M) Overall survival in the shNC and shNUPR1 groups was determined using Kaplan-Meier survival curves. (N) Bioluminescence values for assessing tumor growth in week 4. (O, P) Representative IHC images and corresponding quantitative analysis of NUPR1, MKI67, p62, and LC3B expression in tumor tissues across the treatment groups (scale bars: 100 μm). Data are presented as mean ± SD. Significance levels between specified treatment groups are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cell lysates were first incubated overnight at 4 °C with antibodies against NUPR1 (15056-1-AP, Proteintech), P300 (Santa Cruz Biotechnology, SC-48343), HDAC2 (Santa Cruz Biotechnology, SC-9959), VPS33A (Proteintech, 16896-1-AP), H4K8la (PTM-1415, PTM BIO, 1:1000) or control IgG (Rabbit IgG: 30000-0-AP, Proteintech; Mouse IgG, B900620, Proteintech).

Techniques: Expressing, Activity Assay, Knockdown, Staining, Migration, Luciferase, Imaging

Journal: Theranostics

Article Title: Histone H4K8 lactylation promotes glioblastoma progression by inducing NUPR1-mediated autophagosome‒lysosome fusion

doi: 10.7150/thno.126579

Figure Lengend Snippet: NUPR1 knockdown induces autophagosome accumulation by impairing autolysosome formation. (A) Differences in autophagy pathway scores between cells with high and low NUPR1 expression. (B) TEM images and statistical results showing the changes in the number of autophagosomes in GBM#P3 cells after oxamate treatment (scale bars: 3 μm). (C) Western blot analysis of LC3B and p62 expression in NUPR1-knockdown LN229 and GBM#P3 cells with quantification of grayscale values. (D) Western blot analysis of H4K8la, NUPR1, LC3B and p62 expression after treatment with different concentrations of oxamate. (E-G) TEM images and statistical results of the autophagosomes in LN229 and GBM#P3 cells after siNC and siNUPR1 treatment (scale bars: 3 μm). (H) Fluorescence images of LN229 and GBM#P3 cells transfected with the mCherry-EGFP-LC3B reporter. Cells were treated with siNC or siNUPR1 (scale bars: 10 μm). (I) Fluorescence images of GFP-LC3B and LysoTracker Red in LN229 and GBM#P3 cells treated with siNUPR1 or siNC (scale bars: 10 μm). (J) Statistical results of the ratio of autophagosomes to autolysosomes (yellow puncta/red puncta). (K) Analysis of the colocalization of lysosomes and autophagosomes in GBM#P3 cells. Data are presented as mean ± SD. Significance levels between specified treatment groups are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cell lysates were first incubated overnight at 4 °C with antibodies against NUPR1 (15056-1-AP, Proteintech), P300 (Santa Cruz Biotechnology, SC-48343), HDAC2 (Santa Cruz Biotechnology, SC-9959), VPS33A (Proteintech, 16896-1-AP), H4K8la (PTM-1415, PTM BIO, 1:1000) or control IgG (Rabbit IgG: 30000-0-AP, Proteintech; Mouse IgG, B900620, Proteintech).

Techniques: Knockdown, Expressing, Western Blot, Fluorescence, Transfection

Journal: Theranostics

Article Title: Histone H4K8 lactylation promotes glioblastoma progression by inducing NUPR1-mediated autophagosome‒lysosome fusion

doi: 10.7150/thno.126579

Figure Lengend Snippet: NUPR1 deficiency blocks late-stage autophagy by inhibiting autophagosome‒lysosome fusion. (A) Representative TEM images and quantitative analysis of autophagosome count in GBM#P3 cells under normal conditions and upon BafA1 treatment with NUPR1 knockdown (scale bars: 3 μm). (B-D) Western blot analysis of LC3B and p62 in NUPR1-knockdown cells treated with autophagy modulators (BafA1, 3-MA, Rapa). (E) Confocal microscopy analysis of fluorescence images of LN229 and GBM#P3 cells transfected with the mCherry-EGFP-LC3B reporter in cells treated with siNUPR1 and BafA1, 3-MA, or Rapa and the statistical results of the numbers of autophagosomes and autolysosomes (scale bars: 10 μm). (F) Representative fluorescence images and quantification of GFP-LC3B/LysoTracker Red colocalization in LN229 and GBM#P3 cells following treatment with siNUPR1, Baf, 3-MA, or Rapa (scale bars: 10 μm). Statistical results of the number of colocalized puncta of GFP-LC3B and LysoTracker. Data are presented as mean ± SD. Significance levels between specified treatment groups are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cell lysates were first incubated overnight at 4 °C with antibodies against NUPR1 (15056-1-AP, Proteintech), P300 (Santa Cruz Biotechnology, SC-48343), HDAC2 (Santa Cruz Biotechnology, SC-9959), VPS33A (Proteintech, 16896-1-AP), H4K8la (PTM-1415, PTM BIO, 1:1000) or control IgG (Rabbit IgG: 30000-0-AP, Proteintech; Mouse IgG, B900620, Proteintech).

Techniques: Knockdown, Western Blot, Confocal Microscopy, Fluorescence, Transfection

Journal: Theranostics

Article Title: Histone H4K8 lactylation promotes glioblastoma progression by inducing NUPR1-mediated autophagosome‒lysosome fusion

doi: 10.7150/thno.126579

Figure Lengend Snippet: The NUPR1 inhibitor ZZW-115 blocks autophagy and inhibits GBM progression in vitro . (A) Molecular structure and molecular docking predictions of ZZW-115. (B) IC50 curves showing the response of GBM#P3 and LN229 cell lines to ZZW-115 treatment. (C) Western blot showing the NUPR1, LC3B and p62 levels in LN229 and GBM#P3 cells after treatment with ZZW-115. (D) Representative images and quantitative analysis of the Transwell invasion assays showing the invasive capacity of LN229 cells treated with 3 μM and 6 μM ZZW-115 (scale bars: 200 μm). (E) The effects of 3 μM and 6 μM ZZW-115 on the proliferation of LN229 cells were determined via an EdU staining assay with images and quantitative analysis (scale bars: 100 μm). (F) 3D spheroid invasion assay and statistical analysis of the inhibitory effect of ZZW-115 on long-distance invasion (scale bars: 300 μm). (G) TEM images showing autophagosome accumulation after ZZW-115 treatment and the statistical results of the number of autophagosomes (scale bars: 6 μm). (H) Fluorescence images of LN229 and GBM#P3 cells treated with ZZW-115 and PBS transfected with the mCherry-EGFP-LC3B reporter and analysis of the number of autophagosomes and autolysosomes (scale bars: 10 μm). (I) Fluorescence images and analysis of the colocalization of GFP-LC3B and LysoTracker Red in LN229 and GBM#P3 cells treated with ZZW-115 and PBS (scale bars: 10 μm). (J) Following intracranial implantation of luciferase-expressing GBM#P3 cells, mice were treated with PBS, 3 μM, or 6 μM ZZW-115. Tumor growth was monitored via IVIS imaging, and bioluminescence signals were measured at the indicated time points. (K). Overall survival was analyzed by Kaplan-Meier curves and compared using the log-rank test. (L). Bioluminescence values for assessing tumor growth on day 28. Data are presented as mean ± SD. Significance levels between specified treatment groups are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cell lysates were first incubated overnight at 4 °C with antibodies against NUPR1 (15056-1-AP, Proteintech), P300 (Santa Cruz Biotechnology, SC-48343), HDAC2 (Santa Cruz Biotechnology, SC-9959), VPS33A (Proteintech, 16896-1-AP), H4K8la (PTM-1415, PTM BIO, 1:1000) or control IgG (Rabbit IgG: 30000-0-AP, Proteintech; Mouse IgG, B900620, Proteintech).

Techniques: In Vitro, Western Blot, Staining, Invasion Assay, Fluorescence, Transfection, Luciferase, Expressing, Imaging

Journal: Theranostics

Article Title: Histone H4K8 lactylation promotes glioblastoma progression by inducing NUPR1-mediated autophagosome‒lysosome fusion

doi: 10.7150/thno.126579

Figure Lengend Snippet: ZZW-115 suppresses tumor growth and extends survival by targeting NUPR1 in an orthotopic GBM model. (A-D) IHC staining images of MKi67, NUPR1, LC3B and p62 in tumor tissues across treatment groups, with statistical results of staining scores in the PBS, 3 μm ZZW-115 and 6 μm ZZW-115 groups (scale bars: 100 μm). (E) Study flowchart. Data are presented as mean ± SD. Significance levels between specified treatment groups are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cell lysates were first incubated overnight at 4 °C with antibodies against NUPR1 (15056-1-AP, Proteintech), P300 (Santa Cruz Biotechnology, SC-48343), HDAC2 (Santa Cruz Biotechnology, SC-9959), VPS33A (Proteintech, 16896-1-AP), H4K8la (PTM-1415, PTM BIO, 1:1000) or control IgG (Rabbit IgG: 30000-0-AP, Proteintech; Mouse IgG, B900620, Proteintech).

Techniques: Immunohistochemistry, Staining

Journal: BMC Cancer

Article Title: Integrated bioinformatics analysis of differences between EAC and ESCC

doi: 10.1186/s12885-025-15090-z

Figure Lengend Snippet: Identification and validation of prognostic genes. A Screening of prognostic genes based on Lasso cox regression. B Multivariate Cox analysis showed the prognostic value of six genes in EAC. C Kaplan–Meier curves between patients with high and low riskscore based on the six genes in EAC. D AUC values of the riskscore on 1-, 3-, and 5-year OS in EAC. E Kaplan–Meier curves showed the effect of TUSC3 on OS in ESCC. F RT-qPCR detected the mRNA expression between normal and tumor tissues in ESCC. E Western blot showed the protein expression between normal and tumor tissues in ESCC

Article Snippet: The membrane was subsequently incubated with TUSC3 (1:1000, Proteintech, 16039–1-AP) or GAPDH (1:10000, Proteintech, 60004–1-Ig) overnight at 4 °C.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: A. Lower TUSC4 expression levels was found in both luminal and basal types of breast cancer cell lines, while non-transformed breast cell lines (HMEC, MCF-10A and MCF-12A) exhibited higher TUSC4 level.

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Expressing, Transformation Assay

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: A. TUSC4 knockdown reduced BRCA1 foci formation after IR, while control cells didn't have such effect (NT). γ-H2AX indicated the efficiency of irradiation.

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Knockdown, Control, Irradiation

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: A. Knockdown of TUSC4 didn't significantly change the cell cycle distribution of U2OS cells compared to control cells. G1, G2/M and S phases cells were indicated by percentage of total cell numbers.

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Knockdown, Control

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: A. TUSC4 knockdown increased ubiquitination level of BRCA1 compared to control cells (After MG132 enrichment for ubiquitination).

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Knockdown, Ubiquitin Proteomics, Control

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: A. Colony formation assay indicated that normal U2OS cells are not sensitive to PARP inhibitor Olaparib (1μm) but TUSC4-knockdown cells exhibited increased sensitivity to Olaparib, the colonies were significantly reduced after the treatment (p<0.05).

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Colony Assay, Knockdown

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: Tumorigenicity of Orthotopically Implanted Control and TUSC4-overexpression MDA-MB-231 cells 5×10 6 cells from MDA-AB-231 control and two independent TUSC4-overexpressing MDA-AB-231 cell lines (TUSC4 #7 and TUSC4 #13) were injected per mouse into mammary tumor sizes were analyzed.

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Control, Injection

Journal: Cancer research

Article Title: TUSC4 functions as a tumor suppressor by regulating BRCA1's stability via the E3 ubiquitination pathway

doi: 10.1158/0008-5472.CAN-14-2315

Figure Lengend Snippet: Tumorigenicity of Orthotopically Implanted Control and TUSC4-Knockdown MCF10A cells 1×10 7 cells from MCF-10A control and two independent TUSC4-knockdown MCF-10A cell lines (TUSC4 #1 and TUSC4 #4) were injected per mouse into mammary fat pads glands of 6-week-old female nude mice. Each cell line was injected in five different mice, and tumor sizes were analyzed.

Article Snippet: An anti-TUSC4 antibody was purchased from Proteintech (10157-1-AP), an anti-BRCA1 antibody was purchased from Santa Cruz Biotechnology (sc-6954), and anti-Flag M2 (F3165) and anti-β-actin (A2066) antibodies were purchased from Sigma.

Techniques: Control, Injection

Journal: Molecular Oncology

Article Title: Interaction of BACH2 with FUS promotes malignant progression of glioma cells via the TSLNC8–miR‐10b‐5p–WWC3 pathway

doi: 10.1002/1878-0261.12795

Figure Lengend Snippet: The endogenous expression of BACH2 and FUS, and their effects on the biological behaviour of glioma cells. (A) The expression of BCACH2 was measured by western blotting in NBTs and glioma tissues of grade Ⅰ–Ⅱ and grade Ⅲ–Ⅳ. Data are presented as mean ± SD ( n = 12 for each group) and analysed by using one‐way ANOVA, ** P < 0.01 vs. NBT group; # P < 0.05 vs. Grade Ⅰ‐Ⅱ group. (B) The expression of BACH2 was measured by western blotting in normal HA and glioblastoma cell lines (U87 and U251). Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. HA. (C) The expression of FUS was measured by western blotting in NBTs and glioma tissues of grade Ⅰ–Ⅱ and grade Ⅲ–Ⅳ. Data are presented as mean ± SD ( n = 12 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. NBT group; ## P < 0.01 vs. grade Ⅰ‐Ⅱ group. (D) The expression of FUS was measured by western blotting in normal HA, U87 and U251 cells. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05 vs. HA; ** P < 0.01 vs. HA. (E) CCK‐8 assay was used to measure the effect of BACH2 and FUS on the viability of U87 and U251 cells. (F) Transwell assays were used to measure the effect of BACH2 and FUS on cell migration and invasion of U87 and U251 cells. (G) Flow cytometry analysis of U87 and U251 cells treated with altered expressions of BACH2 and FUS. (E‐G) Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05 vs. sh‐NC group; ** P < 0.01 vs. sh‐NC group; # P < 0.05 vs. sh‐NC group; ## P < 0.01 vs. sh‐NC group; ^^ P < 0.01 vs. sh‐NC+sh‐NC group; ψ P < 0.05 vs. sh‐BACH2 group; ψψ P < 0.01 vs. sh‐BACH2 group; & P < 0.05 vs. sh‐FUS group; && P < 0.01 vs. sh‐FUS group. Scale bar represents 40 μm.

Article Snippet: The primary antibodies were diluted as follows: BACH2 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA), FUS (1 : 1000) (ProteinTech, Rosemont, IL, USA), WWC3 (1 : 100) (Abcam, Cambridge, UK), Yes‐activated protein (YAP) (1 : 1000) (ProteinTech), p‐YAP (1 : 500) (ABclonal Technology, Wuhan, China), GAPDH (1 : 10 000) (ProteinTech) and Histone H3 (1 : 2000) (ProteinTech).

Techniques: Expressing, Western Blot, CCK-8 Assay, Migration, Flow Cytometry

Journal: Molecular Oncology

Article Title: Interaction of BACH2 with FUS promotes malignant progression of glioma cells via the TSLNC8–miR‐10b‐5p–WWC3 pathway

doi: 10.1002/1878-0261.12795

Figure Lengend Snippet: Colocalisation and interaction between BACH2 and FUS, the endogenous expression of TSLNC8 and its effects on the biological behaviour of glioma cells. (A) IF microscopy images of the cellular colocalisation of BACH2 and FUS proteins in U87 and U251 cells ( n = 1). Scale bar represents 40 μm. (B) GST pulldown was used to determine the interaction between BACH2 and FUS in vitro ( n = 1). (C) Glioma cells were subjected to immunoprecipitation using anti‐IgG or anti‐BACH2, followed by immunoblotting with anti‐FUS and anti‐BACH2 ( n = 1). (D) qRT–PCR was used to measure the expression of TSLNC8 in NBTs and glioma tissues of grade Ⅰ–Ⅱ and grade Ⅲ–Ⅳ. Data are presented as mean ± SD ( n = 12 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. NBT group; ## P < 0.01 vs. grade Ⅰ–Ⅱ group. (E) Expression levels of TSLNC8 in normal HA, U87 and U251 cells. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. HA group. (F) FISH microscopy images of the cellular distribution of TSLNC8 in normal HA, U87 and U251 cells ( n = 1). Scale bar represents 20 μm. (G) CCK‐8 assay was used to measure the effect of TSLNC8 on the viability of U87 and U251 cells. (H) Transwell assays were used to measure the effect of TSLNC8 on cell migration and invasion of U87 and U251 cells. (I) Flow cytometry analysis of U87 and U251 cells treated with altered expression of TSLNC8. (G–I) Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. EV group. Scale bar represents 40 μm.

Article Snippet: The primary antibodies were diluted as follows: BACH2 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA), FUS (1 : 1000) (ProteinTech, Rosemont, IL, USA), WWC3 (1 : 100) (Abcam, Cambridge, UK), Yes‐activated protein (YAP) (1 : 1000) (ProteinTech), p‐YAP (1 : 500) (ABclonal Technology, Wuhan, China), GAPDH (1 : 10 000) (ProteinTech) and Histone H3 (1 : 2000) (ProteinTech).

Techniques: Expressing, Microscopy, In Vitro, Immunoprecipitation, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Migration, Flow Cytometry

Journal: Molecular Oncology

Article Title: Interaction of BACH2 with FUS promotes malignant progression of glioma cells via the TSLNC8–miR‐10b‐5p–WWC3 pathway

doi: 10.1002/1878-0261.12795

Figure Lengend Snippet: Endogenous expression of miR‐10b‐5p and its effects on the biological behaviour of glioma cells. (A) qRT–PCR was used to measure the expression of TSLNC8 in U87 and U251 cells, which were treated by knockdown of BACH2 and FUS. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05 vs. sh‐NC group; ** P < 0.01 vs. sh‐NC group; ## P < 0.01 vs. sh‐NC group; ^^ P < 0.01 vs. sh‐NC+sh‐NC group; ψψ P < 0.01 vs. sh‐BACH2 group; && P < 0.01 vs. sh‐FUS group. (B) BACH2 binds to the promoter of TSLNC8 in U87 and U251 cells. A schematic representation of the human BACH2 promoter region 3000‐bp upstream of the TSS, which was designated as +1. Putative BACH2‐binding sites are illustrated. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a NC ( n = 1). (C) qRT–PCR was used to measure the expression of miR‐10b‐5p in NBTs and glioma tissues of grade I–II and grade III–IV. Data are presented as mean ± SD ( n = 12 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. NBT group; ## P < 0.01 vs. grade Ⅰ–Ⅱ group. (D) Expression levels of miR‐10b‐5p in normal HA, U87 and U251 cells. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. HA group. (E) The CCK‐8 assay was used to measure the effect of miR‐10b‐5p on the proliferation of U87 and U251 cells. (F) Transwell assays were used to measure the effect of miR‐10b‐5p on the migration and invasion of U87 and U251 cells. (G) Flow cytometry analysis of U87 and U251 cells treated with altered expression of miR‐10b‐5p. (E–G) Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05 vs. agomir‐10b‐5p‐NC group; ** P < 0.01 vs. agomir‐10b‐5p‐NC group; # P < 0.05 vs. antagomir‐10b‐5p‐NC group, ## P < 0.01 vs. antagomir‐10b‐5p‐NC group. Scale bar represents 40 μm.

Article Snippet: The primary antibodies were diluted as follows: BACH2 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA), FUS (1 : 1000) (ProteinTech, Rosemont, IL, USA), WWC3 (1 : 100) (Abcam, Cambridge, UK), Yes‐activated protein (YAP) (1 : 1000) (ProteinTech), p‐YAP (1 : 500) (ABclonal Technology, Wuhan, China), GAPDH (1 : 10 000) (ProteinTech) and Histone H3 (1 : 2000) (ProteinTech).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Binding Assay, Immunoprecipitation, Amplification, CCK-8 Assay, Migration, Flow Cytometry

Journal: Molecular Oncology

Article Title: Interaction of BACH2 with FUS promotes malignant progression of glioma cells via the TSLNC8–miR‐10b‐5p–WWC3 pathway

doi: 10.1002/1878-0261.12795

Figure Lengend Snippet: BACH2, FUS, TSLNC8 and miR‐10b‐5p regulated WWC3 expression and YAP phosphorylation levels. (A, B) Western blotting assay was used to measure WWC3 expression and p‐YAP levels in U87 and U251 cells treated with miR‐10b‐5p overexpression or knockdown. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05, ** P < 0.01 vs. agomir‐10b‐5p‐NC group; ## P < 0.01 vs. antagomir‐10b‐5p‐NC group. (C) Western blotting assay was used to measure the p‐YAP level in U87 and U251 cells, which are regulated by miR10b‐5p targeting WWC3 3′‐UTR. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05, ** P < 0.01 vs. miR‐10b‐5p‐NC+WWC3‐NC group; # P < 0.05 vs. miR‐10b‐5p+WWC3‐NC group; ^^ P < 0.01 vs. miR‐10b‐5p+WWC3 group. (D) Western blotting assay was used to measure the cytoplasmic YAP expressions in U87 and U251 cells, which are regulated by miR10b‐5p targeting WWC3 3′‐UTR. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05, ** P < 0.01 vs. miR‐10b‐5p‐NC+WWC3‐NC group; # P < 0.05, ## P < 0.01 vs. miR‐10b‐5p+WWC3‐NC group; ^^ P < 0.01 vs. miR‐10b‐5p+WWC3 group. (E) Western blotting assay was used to measure the nuclear YAP expressions in U87 and U251 cells, which are regulated by miR10b‐5p targeting WWC3 3′‐UTR. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.05 vs. miR‐10b‐5p‐NC+WWC3‐NC group; ## P < 0.01 vs. miR‐10b‐5p+WWC3‐NC group; ^ P < 0.05, ^^ P < 0.01 vs. miR‐10b‐5p+WWC3 group. (F, G) Western blotting assay was used to measure WWC3 expressions and p‐YAP levels in U87 and U251 cells, which were treated by knockdown of BACH2 and FUS. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. * P < 0.05, ** P < 0.01 vs. sh‐NC group; # P < 0.05, ## P < 0.01 vs. sh‐NC group; ^^ P < 0.01 vs. sh‐NC+sh‐NC group; ψψ P < 0.01 vs. sh‐BACH2 group; && P < 0.01 vs. sh‐FUS group. (H, I) Western blotting assay was used to measure WWC3 expressions and p‐YAP levels in U87 and U251 cells, which were treated with overexpressed TSLNC8. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. EV group. (J, K) Western blotting assay was used to measure WWC3 expressions and p‐YAP levels regulated by TSLNC8 and miR10b‐5p in U87 and U251 cells. Data are presented as mean ± SD ( n = 3 for each group) and analysed by using one‐way ANOVA. ** P < 0.01 vs. EV+antagomir‐10b‐5p ‐NC group.

Article Snippet: The primary antibodies were diluted as follows: BACH2 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA), FUS (1 : 1000) (ProteinTech, Rosemont, IL, USA), WWC3 (1 : 100) (Abcam, Cambridge, UK), Yes‐activated protein (YAP) (1 : 1000) (ProteinTech), p‐YAP (1 : 500) (ABclonal Technology, Wuhan, China), GAPDH (1 : 10 000) (ProteinTech) and Histone H3 (1 : 2000) (ProteinTech).

Techniques: Expressing, Phospho-proteomics, Western Blot, Over Expression, Knockdown

Journal: Molecular Oncology

Article Title: Interaction of BACH2 with FUS promotes malignant progression of glioma cells via the TSLNC8–miR‐10b‐5p–WWC3 pathway

doi: 10.1002/1878-0261.12795

Figure Lengend Snippet: Tumour xenograft studies. (A) The nude mice carrying tumours from the respective groups are shown. The sample tumours excised from the respective groups are shown. (B) Tumour growth curves of six nude mice groups are shown, and data are presented as mean ± SD ( n = 8 for each group). Tumour volume was calculated every 5 days after injection, and the tumour was excised after 45 days. Data are analysed by using one‐way ANOVA * P < 0.05, ** P < 0.01 vs. control group; # P < 0.05 vs. sh‐BACH2 group; ^ P < 0.05 vs. sh‐FUS group; ψ P < 0.05 vs. TSLNC8‐OE group. (C) Survival curves of nude mice injected into the right striatum from the respective groups are shown ( n = 8, each group). Data are analysed by using log‐rank test, P < 0.05 for sh‐BACH2, sh‐FUS or TSLNC8‐OE group vs. control group; P < 0.01 for sh‐BACH2+sh‐FUS+TSLNC8 group‐OE vs. control group. (D) Schematic drawing of the mechanism of the BACH2/TSLNC8/miR‐10b‐5p/wwc3 axis in glioma cells.

Article Snippet: The primary antibodies were diluted as follows: BACH2 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA), FUS (1 : 1000) (ProteinTech, Rosemont, IL, USA), WWC3 (1 : 100) (Abcam, Cambridge, UK), Yes‐activated protein (YAP) (1 : 1000) (ProteinTech), p‐YAP (1 : 500) (ABclonal Technology, Wuhan, China), GAPDH (1 : 10 000) (ProteinTech) and Histone H3 (1 : 2000) (ProteinTech).

Techniques: Injection, Control

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Molecular expression levels of TMEM16A in LV. (A) TMEM16A mRNA expression level in LV ( n = 3 mice in each group per time point, RM two-way ANOVA). (B) Traditional western blot results of TMEM16A protein expression level in LV ( n = 3 mice in each group per time point, RM two-way ANOVA). (C) Simple western blot results of TMEM16A protein expression level in LV ( n = 3 mice in each group per time point, RM two-way ANOVA).

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Expressing, Western Blot, Simple Western

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: I to in LVMs. (A) Recordings show the effects of T16A inh -A01 (30 μM) or 4-AP (5 mM) on I to in LVMs. (B) Recordings show the effects of Anti-TMEM16A antibody (1:200) or 4-AP (5 mM) on I to in LVMs. (C) I-V relations of T16A inh -A01 sensitive current and Anti-TMEM16A antibody sensitive current ( n = 4 cells in each group, two-way ANOVA). (D) I-V relation of 4-AP sensitive current ( n = 4 cells in each group, two-way ANOVA).

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques:

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: I TMEM16A in LVMs. (A) Superimposed current tracings were recorded from the same cell before (control) and after exposure to 30 μM T16A inh -A01. Right panel shows expanded traces in the blue box of left panel. (B) Bar graphs show average T16A inh -A01 sensitive current densities in LVMs ( n = 7—8 cells from three mice in each group per time point, RM two-way ANOVA).

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Control

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Molecular expression levels of TMEM16A in HUVECs. (A) TMEM16A mRNA expression level in HUVECs (n = 3–4). (B) Western blot results of TMEM16A protein expression level in HUVECs ( n = 3–5). * p < 0.05, ** p < 0.01 and *** p < 0.001, one-way ANOVA and unpaired t -test.

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Effect of TMEM16A on migration in HUVECs. (A) Wound healing assay results of HUVECs transfected with siRNA NC or siRNA Mix , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately ( n = 5—8 in each group per time point). (B) Wound healing assay results of HUVECs transfected with TM NC or TM OE , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately (n = 5 – 7 in each group per time point). * p < 0.05 and ** p < 0.01 compared with siRNA NC or TM NC under the same treatment and at the same time point, # p < 0.05 and ## p < 0.01 compared with vehicle (PBS) in the same cell type and at the same time point, RM two-way ANOVA.

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Migration, Wound Healing Assay, Transfection

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Effect of TMEM16A on angiogenesis in HUVECs. (A,C) Tube formation assay results of HUVECs transfected with siRNA NC or siRNA Mix , or TM NC , or TM OE , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately (n = 3–9). (B,D) Endothelial cell spheroids sprouting assay results of HUVECs transfected with siRNA NC or siRNA Mix , or TM NC , or TM OE , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately ( n = 3–5). * p < 0.05 compared with siRNA NC or TM NC under the same treatment, # p < 0.05 and ## p < 0.01 compared with vehicle (PBS) in the same cell type, two-way ANOVA.

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Tube Formation Assay, Transfection

Journal: International Journal of Peptide Research and Therapeutics

Article Title: Peptide-Based Vaccines and Therapeutics for COVID-19

doi: 10.1007/s10989-022-10397-y

Figure Lengend Snippet: A review on COVID-19 peptide-based vaccines: companies

Article Snippet: Axon neuroscience , ACvac1 , Pluri-epitope peptide vaccine , Together against Alzheimer’s disease ( ) .

Techniques: Vaccines