Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: ERBB2 expression in different molecular types of MIBC. (A) Schematic diagram of consensus types of MIBC, including basal/squamous (Ba/Sq), luminal unstable (LumU), luminal nonspecified (LumNS), luminal papillary (LumP), neuroendocrine-like (NE-like), and stoma-rich. (B) ERBB2 mRNA expression in six MIBC consensus subtypes in the TCGA-MIBC dataset. (C) ERBB2 mRNA expression in luminal and basal cells in CCLE dataset. (D) The luminal and basal-type single-cell clusters were determined by their molecular markers (luminal: FOXA1 and GATA3; basal: CD44 and KRT5), and the distribution of ERBB2 mRNA expression in luminal and basal clusters is shown. (E) The luminal and basal bladder cancer tissues’ IHC were classified by their molecular marker (luminal: KRT20 and GATA3; basal: CD44 and KRT5/6), and ERBB2 protein expression was detected. (F) Quantification of ERBB2 immunohistochemistry protein levels in luminal and basal bladder cancer. (G) Western blot analysis showed the protein expression of ERBB2, KRT20, GATA3, CD44, and KRT5/6 in luminal (HT1376, RT4, and SW780) and basal (5637, J82, and T24) cells. (H) Quantitative chart of ERBB2, KRT20, GATA3, CD44, and KRT5/6 from the Western blot analysis. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.

Article Snippet: The ERBB2 overexpression lentivirus vectors were purchased from Genechem (Shanghai, China).

Techniques: Expressing, Marker, Immunohistochemistry, Western Blot

Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: Luminal cells show higher sensitivity to RC48. (A) The Western blot shows ERBB2 protein expression levels in luminal cells (SW780, HT1376, and RT4) after exposure to RC48 at different concentrations and times. (B) The barplot shows the gDS in MIBC cells representing luminal and basal cells. (C) The percentage of surviving cells after exposing different concentrations of RC48 in three luminal (SW780, RT4, and HT1376) and three basal (J82, T24, and 5637) cells. (D) Colon formation assay reveals the cell growth after exposing different concentrations of RC48 in luminal and basal cells. The percentage of survival cells was quantified. (E) Cell viability of three luminal and three basal cells after exposing RC48 (25 μg/ml) at different times. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001; ns, not significant.

Article Snippet: The ERBB2 overexpression lentivirus vectors were purchased from Genechem (Shanghai, China).

Techniques: Western Blot, Expressing, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: ERBB2 expression mediates the sensitivity of luminal and basal cells to RC48. (A) Western blot analysis validated the ERBB2 expression in ERBB2-silenced luminal cells (SW780 and HT1376) and ERBB2-overexpressing basal (T24 and 5637) cells. (B , C) The percentage of surviving cells after exposure to different concentrations of RC48 in ERBB2-silenced luminal cells (SW780 and HT1376)/or ERBB2-overexpressing basal cells (T24 and 5637). (D) Colon formation assay demonstrates cell growth after exposure to RC48 (5 μg/ml) in ERBB2-overexpressing basal cells. (E , F) Cell viability of the ERBB2-overexpressing and control basal cells after exposure to RC48 (25 μg/ml). (G) Tumors with different treatments were shown. (H) Tumor weight was measured after 4 weeks. Data are presented as the mean ± SD. (I) Tumor volume was measured one to two times weekly once the tumor became visible. Data are presented as the mean ± SD. (J) IHC revealed ERBB2 expression in tumor samples from mice receiving different treatments. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.

Article Snippet: The ERBB2 overexpression lentivirus vectors were purchased from Genechem (Shanghai, China).

Techniques: Expressing, Western Blot, Tube Formation Assay, Control

Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: The STAT3 pathway is compensatively activated in basal cells. (A) The diagram shows the GESA result based on the DEGs between basal/squamous and the other three luminal consensus subtypes. (B) The Venn diagram shows the common altered pathways from the GSEA results based on the DEGs determined between basal and luminal types of four molecular classification systems. (C, D) The heatmap shows the expression of genes positively correlated with STAT3 pathways in basal and luminal bladder cancer tissues and cell lines. (E) STAT3 mRNA expression levels in consensus types of TCGA-MIBC. (F) STAT3 mRNA expression levels in luminal and basal cells. (G) STAT3 protein levels in luminal and basal cells were detected by Western blot analysis. (H) The diagram shows the GSEA results based on the DEGs between RC48-exposed SW780 and control. The top 10 activated pathways are shown. (I) The diagram shows the correlation between ERBB2 and STAT3 in mRNA levels, with the PCC used for analysis. (J, K) In ERBB2-silenced luminal cells and ERBB2-overexpressing basal cells, Western blot analysis was performed to measure the expression changes of ERBB2, STAT3, and pSTAT3 (Tyr705). (L) After exposure to RC48 (HT1376 and SW780), the proteins ERBB2, STAT3, and pSTAT3 (Tyr705) were detected by Western blot analysis. (M) Control and RC48-exposed SW780 and HT1376 cells, or ERBB2-overexpressing T24 and 5637 cells, were fixed and incubated with antibodies against pSTAT3 (Tyr705) for immunofluorescence analysis. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.

Article Snippet: The ERBB2 overexpression lentivirus vectors were purchased from Genechem (Shanghai, China).

Techniques: Expressing, Western Blot, Control, Incubation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: The combination treatment of RC48 and STAT3 inhibitor acts as a promising therapeutic strategy for basal bladder cancer

doi: 10.3389/fimmu.2024.1432586

Figure Lengend Snippet: The combination of ART and RC48 blocks tumor growth in the xenograft model of nude mice. (A) Schematic diagram of the treatment paradigm. (B) Tumors with different treatments are shown. (C) Tumor weight was measured after 4 weeks. Data are presented as the mean ± SD. (D) Tumor volume was measured one to two times per week when the tumor became visible. Data are presented as the mean ± SD. (E) The Kaplan–Meier survival curve shows the prognostic differences among the control, RC48, artesunate, and RC48+artesunate groups. (F) The diagram shows changes in mouse body weight. (G) IHC shows the STAT3 expression in tumor samples from mice with different treatments. (H) Diagram of STAT3 pathways after treatment with RC48 in luminal and basal cells. (I) A combination of STAT3 and ERBB2 inhibition in vivo and mechanism diagram. *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001; ns, not significant.

Article Snippet: The ERBB2 overexpression lentivirus vectors were purchased from Genechem (Shanghai, China).

Techniques: Control, Expressing, Inhibition, In Vivo

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b in chondrosarcoma cells inhibits cells growth rate and increases the sensitivity to doxorubicin. Notes: ( A ) Transfection of pre-miR-125b into JJ012, CH-2879, and SW1353 cells; the expressions of miR-125b were measured by qPCR. ( B ) Overexpression of miR-125b inhibits the JJ012, CH-2879, and SW1353 cells growth rates. Cells were transfected with pre-miR-125b for 48 hours, and then were plated into 48-well plates for the cell growth assays. ( C ) JJ012, CH-2879, and SW1353 cells were transfected with negative control and pre-miR-125b for 48 hours; the cells were plated into 48-well plates for the treatments with doxorubicin at indicated concentrations for 48 hours, followed by the cell viability assays. Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Transfection, Negative Control

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b in chondrosarcoma cells downregulates glucose metabolism. Notes: ( A ) Glucose uptake and ( B ) lactate product were measured in miR-125b overexpressing JJ012 (left) and CH-2879 (right) cells compared with negative control. ( C ) Western blotting experiments showed that the expressions of HK II, PDK1, and LDHA were downregulated in miR-125b overexpressing JJ012 (left) and CH-2879 (right) cells compared with negative control. β-actin was used as a loading control. Columns, mean of three independent experiments; bars, SE. * P <0.05.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Negative Control, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Restoration of ErbB2 in miR-125b overexpressing cells recovers the glucose metabolism and doxorubicin sensitivity. Notes: ( A ) JJ012 cells were transfected with 100 nM pre-miR-negative control and pre-miR-125b for 48 hours, followed by transfection with vector control and overexpression vector containing wild-type ErbB2 for 24 hours, and then cells were collected and prepared for Western blotting with antibody against ErbB2, HK II, PDK1, and LDHA. β-actin was used as a loading control. ( B ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ), then the glucose uptake (left) and lactate product (right) were checked. ( C ) JJ012 cells were transfected with pre-miR-125b and ErbB2 as described in ( A ); cells were collected and replaced in 48-well plates for overnight, followed by doxorubicin treatments at the indicated concentrations for 48 hours. Cells were analyzed by cell viability assays. ( D ) The JJ012 cells were transfected with control siRNA or siErbB2 for 48 hours, and then cells were collected for Western blot analysis (middle) and the measurements of glucose uptake and lactate product. Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Transfection, Negative Control, Plasmid Preparation, Over Expression, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism

doi: 10.2147/DDDT.S90530

Figure Lengend Snippet: Overexpression of miR-125b resensitizes doxorubicin resistant chondrosarcoma cells through the inhibition of glucose metabolism. Notes: ( A ) Overexpression of miR-125b resensitized doxorubicin resistant cells. Cells were transfected with pre-miR-125b for 48 hours and then were treated with doxorubicin at indicated concentrations for 48 hours, followed by the measurement of cell viability. ( B ) Western blotting experiments showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the expressions of ErbB2, HK II, PDK1, and LDHA to the same levels as those of JJ012 parental cells. ( C ) Glucose uptake (left) and lactate product (right) results showed the overexpression of miR-125b in JJ012 doxorubicin resistant cells decreased the glucose metabolism to the same levels as those of JJ012 parental cells. ( D ) Exogenous overexpression of LDHA into miR-125b pretransfected JJ012 parental cells (left) showed resistant to doxorubicin treatments at the indicated concentrations for 48 hours (right). Columns, mean of three independent experiments; bars, SE. * P <0.05; ** P <0.01. Abbreviation: Doxo R, Doxo resistant.

Article Snippet: Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com .

Techniques: Over Expression, Inhibition, Transfection, Western Blot

Journal: Cell Systems

Article Title: Simultaneous Multiplexed Imaging of mRNA and Proteins with Subcellular Resolution in Breast Cancer Tissue Samples by Mass Cytometry

doi: 10.1016/j.cels.2017.12.001

Figure Lengend Snippet:

Article Snippet: ERBB2 overexpression vector , Addgene , Plasmid #23888.

Techniques: Microarray, Recombinant, Labeling, Multiplex Assay, Purification, Expressing, Over Expression, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Destabilized 3’UTR ARE therapeutically degrades ERBB2 in drug-resistant ERBB2+ cancer models

doi: 10.1101/2022.08.14.503914

Figure Lengend Snippet: A. Dot blots show the phospho kinase array pattern of the BT474 clone 5 wild type, BT474 clone 5 Vector, BT474 clone 5 desARE3’UTR ERBB2-3 and BT474 clone 5 desARE3’UTR c-MYC 2-3. The dot marked in red box show unique kinases down regulated only in BT474 clone 5 desARE3’UTR ERBB2-3 and the dot marked in green boxes show unique kinases downregulated only in BT474 clone 5 desARE3’UTR c-MYC 2-3. Each kinase is spotted twice. B. The bar chart shows the intensity of the phospho-kinase array dot blot for the BT474 clone 5 wild type (black bar), BT474 clone 5 Vector (blue bar), BT474 clone 5 desARE3’UTR ERBB2-3 (red bar) and BT474 clone 5 desARE3’UTR c-MYC 2-3 (green bar). C. The bar charts show YES1 pT60 and WNK1 pT60 kinases specifically down regulated in BT474 clone 5 desARE3’UTR ERBB2-3 in red bar. Controls BT474 clone 5 wild type (black bar), BT474 clone 5 Vector (blue bar), and BT474 clone 5 desARE3’UTR c-MYC 2-3 (green bar). T-test (*** Pval=0.00413, desARE3’UTRERBB2-3 for YES and WNK1) D. Venn diagram show kinases downregulated in destabilized desARE3’UTR ERBB2-3, 30 NCI-H1975 (p=0.02) blue (from RNA Seq), and in destabilized desARE3’UTR ERBB2-3 BT474 clone 5 (red-kinase array) and the shared kinases found downregulated in destabilized ERBB2 in both resistant cell lines WNK1 and YES1 in pink. E. Western blot show YES1 and GAPDH expression across the NCI-H1975 wildtype cells, vector, cells containing constructs desARE3’UTR ERBB2-1, 3 and 30 and wild type trastuzumab treated NCI-H1975 cells (n=2). F. Bar chart show quantification of the YES1 expression normalized against GAPDH on NCI-H1975 wildtype cells, vector and cells containing constructs desARE3’UTR ERBB2-1, 3 and 30 (n=2). T-test (***Pval=0.0021, desARE3’UTRERBB2-1,3,30) G. Western blot show WNK1 and GAPDH expression across the NCI-H1975 wildtype cells, vector, cells containing constructs desARE3’UTR ERBB2-1, 3 and 30 and wild type trastuzumab treated NCI-H1975 cells. H. Western blot show YES1 and GAPDH expression across the NCI-H1975 destabilized with constructs desARE3’UTR ERBB2-3 and 30 (lane 1 and 3) and over expressed with YES1 in lane 2 and 4. I. Bar chart show the wound healing closing of NCI H1975 wildtype, vector, desARE3’UTRERBB2-1, 3,30 and desARE3’UTR ERBB2-30,30 overexpressed with YES1 from 0hr to 72hrs. At 24hrs *** p=0.00025 desARE3’UTRERBB2-3,30. At 48hrs ****p=0.000018 desARE3’UTR ERBB2-3 and at 72hrs *** p=0.0003 desARE3’UTRERBB2-3 and **p=0.002 desARE3’UTRERBB2-30.

Article Snippet: YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and 30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Plasmid Preparation, Dot Blot, RNA Sequencing, Western Blot, Expressing, Construct

Journal: bioRxiv

Article Title: Destabilized 3’UTR ARE therapeutically degrades ERBB2 in drug-resistant ERBB2+ cancer models

doi: 10.1101/2022.08.14.503914

Figure Lengend Snippet: A. Microscope images of wound healing assay of NCI H1975 WT, vector, desARE3’UTRERBB2-1, 3 and 30 and desARE3’UTRERBB2-3 and 30 overexpressed with YES1.Images were taken at 0hr, 24hr, 48hrs and 72hrs at 10x.

Article Snippet: YES1 Y537F overexpression vector (Addgene: 51299) was transfected into desARE3’UTR ERBB2-3 and 30 cells that have lost ERBB2 and YES1 by introduction of destabilized desARE3’UTR ERBB2.

Techniques: Microscopy, Wound Healing Assay, Plasmid Preparation