Journal: Theranostics

Article Title: Single cell analyses reveal the PD-1 blockade response-related immune features in hepatocellular carcinoma

doi: 10.7150/thno.95971

Figure Lengend Snippet: IL1B + cDC2s are the main executor on cDC2s. UMAP plot showing DC subgroups of the discovery cohort. (B) Bubble plot displaying unique markers of different DC subgroups. (C) Differential expression of genes related to DC function across DC subgroups. (D) UMAP plot illustrating the distribution of DC subgroups across different tissues and under different treatment conditions. (E) Bar graph showing the proportion of DC subgroups across different tissues and under different treatment conditions. (F) Volcano plot depicting differential genes between two cDC2 subgroups. (G) Multicolor immunofluorescence staining confirms the presence of two types of cDC2 in human HCC tissues. (H) Bubble plot shows the differences in the co-receptor pairing between the two types of cDC2 and CD4 + T cell subgroups (p-values < 0.05, IL1B + cDC2 vs DPYD + cDC2). (I) Cell interactions between IL1B + cDC2 dendritic cell and CD4 + T cell subgroups in the tumor tissue of non-responsive patients. (J) Cell interactions between DPYD + cDC2 dendritic cell and CD4 + T cell subgroups in the tumor tissue of non-responsive patients. (K) Communication of the CD40 signaling pathway between cDC2 dendritic cells and CD4 + T cell subgroups. (L) Communication of the TGF-β signaling pathway between cDC2 dendritic cells and CD4 + T cell subgroups.

Article Snippet: Related antibodies used in this study included TREM2 (RD, Cat#MAB17291), CD68 (abcam, Cat#ab289671), PanCK (abcam, Cat#ab234297), POSTN (proteintech, Cat#66491-1-lg), CD3 (abcam, Cat#ab16669), CLEC10A (abcam, Cat#ab315086), HSPA1B (proteintech, Cat#10995-1-AP), DPYD (proteintech, Cat#27662-1-AP), IL1B (proteintech, Cat#16806-1-AP) and THEMIS (proteintech, Cat#27415-1-AP).

Techniques: Quantitative Proteomics, Multicolor Immunofluorescence Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ZIP10 drives osteosarcoma proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway

doi: 10.1186/s13046-021-02146-8

Figure Lengend Snippet: ZIP10 increases ITGA10 expression in osteosarcoma. a Gene array analysis of NC and shZIP10 Saos-2 cells. Up−/down-regulated genes associated with the integrin family are shown. b qRT-PCR analysis of integrins in Saos-2. c Expression levels of AKT, p-AKT, PAK, p-PAK, p-FAK and p-SRC were analyzed by WB using 143B and Saos-2 cells with/without ITGA10 knockdown. d WB analysis of AKT and PAK activation with/without ZIP10 overexpression in 143B cells. e Protein levels of p-AKT and p-PAK were quantified using ImageJ software. f A CCK-8 assay was performed to determine cell proliferation. g Flow cytometry was conducted to determine apoptosis. Apoptosis rates were quantified. h The whole-cell extract of 143B cells treated with/without cisplatin (Cis) for 72 h was subjected to WB analysis. Data are means ± SEM; * P < 0.05, ** P < 0.01

Article Snippet: After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies (1:1000) against ACTB (CST; #4970), ZIP10 (SAB; #24824), AKT (CST; #4691), p-AKT (CST; #4060), ERK (CST; #4695), p-ERK (CST; #9101), JNK (CST; #9252), p-JNK (CST; #4671), C-PARP (CST; #5625), C-caspase-3 (CST; #9661), ITGA10 (SAB; #44824), PAK (CST; #2602), p-PAK (CST; #2606), p-FAK (CST; #8556), p-SRC (CST; #12432), CREB (CST; #12432), and p-CREB (CST; #9198) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Activation Assay, Over Expression, Software, CCK-8 Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ZIP10 drives osteosarcoma proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway

doi: 10.1186/s13046-021-02146-8

Figure Lengend Snippet: ZIP10 increases CREB phosphorylation, leading to upregulation of ITGA10 transcription. a , b Zn concentrations were analyzed in OS cells with/without ZIP10 knockdown using FluoZin-3 AM. c , d ITGA10 mRNA ( c ) and protein ( d ) levels were analyzed in OS cells treated with Zn supplementation (ZnSO 4 ) or Zn chelator (TPEN). e WB analysis of CREB and p-CREB expression in 143B cells treated with/without ZnSO 4 . f WB analysis of p-CREB in 143B cells with/without ITGA10 knockdown. WB analysis of ITGA10 in 143B cells with/without CREB inhibition (666–15). g Schematic diagram showing the promoter structures of luciferase constructs: the wild-type ITGA10 promoter (WT), deletion mutants lacking CRE1 (△CRE1), CRE2 (△CRE2), or both (△CRE1 + 2). The CRE1 and CRE2 motif sequences are indicated. TSS, transcription start site. LUC, luciferase. h 143B cells were transfected with plasmids encoding ITGA10 (− 1300/− 800)-Luc WT, △CRE1, △CRE2 or △CRE1 + 2. Then, OS cells were treated with Zn supplementation (ZnSO 4 ) or CREB inhibitor (666–15). Luciferase levels were measured in extracts after 48 h. i ChIP of putative CREB response elements (CRE 1 and 2) within the ITGA10 promoter region determined by PCR. The promoter’s non-CRE region (Ctrl motif) and the β-actin region were used as negative controls; histone antibody or rabbit IgG were assay controls. j ChIp results confirmed by qRT-PCR. Data are means ± SEM; * P < 0.05, ** P < 0.01

Article Snippet: After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies (1:1000) against ACTB (CST; #4970), ZIP10 (SAB; #24824), AKT (CST; #4691), p-AKT (CST; #4060), ERK (CST; #4695), p-ERK (CST; #9101), JNK (CST; #9252), p-JNK (CST; #4671), C-PARP (CST; #5625), C-caspase-3 (CST; #9661), ITGA10 (SAB; #44824), PAK (CST; #2602), p-PAK (CST; #2606), p-FAK (CST; #8556), p-SRC (CST; #12432), CREB (CST; #12432), and p-CREB (CST; #9198) overnight at 4 °C.

Techniques: Phospho-proteomics, Knockdown, Expressing, Inhibition, Luciferase, Construct, Transfection, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ZIP10 drives osteosarcoma proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway

doi: 10.1186/s13046-021-02146-8

Figure Lengend Snippet: ZIP10-ITGA10-p-AKT signaling is required for the maintenance of cisplatin resistance in vivo. a - c Photographed xenograft tumors ( a ), average tumor volume ( b ) and tumor weight ( c ) of 143B-NC, 143B-oeZIP10, 143B-oeZIP10 + 666–15 and 143B-oeZIP10 + GSK690693 xenografts treated with vehicle (Con) or cisplatin (Cis). Scale bar, 1 cm. N = 6 mice per group. d WB analysis was performed to detect target proteins, as indicated in xenograft tissues. e IHC staining analysis of ZIP10, ITGA10, p-AKT, Ki67 and C-caspase-3 in xenograft tissues. Scale bar, 20 μm. Data are means ± SEM; * P < 0.05, ** P < 0.01

Article Snippet: After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies (1:1000) against ACTB (CST; #4970), ZIP10 (SAB; #24824), AKT (CST; #4691), p-AKT (CST; #4060), ERK (CST; #4695), p-ERK (CST; #9101), JNK (CST; #9252), p-JNK (CST; #4671), C-PARP (CST; #5625), C-caspase-3 (CST; #9661), ITGA10 (SAB; #44824), PAK (CST; #2602), p-PAK (CST; #2606), p-FAK (CST; #8556), p-SRC (CST; #12432), CREB (CST; #12432), and p-CREB (CST; #9198) overnight at 4 °C.

Techniques: In Vivo, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ZIP10 drives osteosarcoma proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway

doi: 10.1186/s13046-021-02146-8

Figure Lengend Snippet: ZIP10 correlates positively with p-CREB, ITGA10 and p-AKT in osteosarcoma patients. a Representative cases from 52 OS specimens (without chemotherapy) were analyzed by IHC staining for Ki67, ZIP10, p-CREB, ITGA10 and p-AKT. Scale bar, 20 μm. b - f The expression of ZIP10 and p-CREB ( b ), ZIP10 and ITGA10 ( c ), ZIP10 and p-AKT ( d ), ITGA10 and p-CREB ( e ), and ITGA10 and p-AKT ( f ) was analyzed in 52 OS specimens. The relative proportions of protein expression are illustrated as a pie chart. g - i The percentage of samples displaying low or high ZIP10 expression compared to the expression levels of p-CREB ( g ), ITGA10 ( h ) and p-AKT ( i ). j , k The percentage of specimens displaying low or high ITGA10 expression compared to the expression levels of p-CREB ( j ) and p-AKT ( k ). l - p Scatterplot showing the positive correlation between ZIP10 and p-CREB ( l ), ZIP10 and ITGA10 ( m ), ZIP10 and p-AKT ( n ), ITGA10 and p-CREB ( o ), ITGA10 and p-AKT ( p ) expression in OS patients

Article Snippet: After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies (1:1000) against ACTB (CST; #4970), ZIP10 (SAB; #24824), AKT (CST; #4691), p-AKT (CST; #4060), ERK (CST; #4695), p-ERK (CST; #9101), JNK (CST; #9252), p-JNK (CST; #4671), C-PARP (CST; #5625), C-caspase-3 (CST; #9661), ITGA10 (SAB; #44824), PAK (CST; #2602), p-PAK (CST; #2606), p-FAK (CST; #8556), p-SRC (CST; #12432), CREB (CST; #12432), and p-CREB (CST; #9198) overnight at 4 °C.

Techniques: Immunohistochemistry, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ZIP10 drives osteosarcoma proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway

doi: 10.1186/s13046-021-02146-8

Figure Lengend Snippet: Proposed model of ZIP10 regulation of proliferation and chemoresistance in osteosarcoma. ZIP10 expression is elevated in OS, which might be a result of Zn deficiency and chemotherapy. ZIP10 increases Zn uptake and activates CREB and its binding to the ITGA10 promotor and thus promotes ITGA10 expression in OS cells. ITGA10 is an important membrane-anchored protein that confers OS cell proliferation and chemoresistance through activation of the PI3K/AKT signaling pathway. Targeting ZIP10 in OS might increase chemotherapy sensitization in OS

Article Snippet: After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies (1:1000) against ACTB (CST; #4970), ZIP10 (SAB; #24824), AKT (CST; #4691), p-AKT (CST; #4060), ERK (CST; #4695), p-ERK (CST; #9101), JNK (CST; #9252), p-JNK (CST; #4671), C-PARP (CST; #5625), C-caspase-3 (CST; #9661), ITGA10 (SAB; #44824), PAK (CST; #2602), p-PAK (CST; #2606), p-FAK (CST; #8556), p-SRC (CST; #12432), CREB (CST; #12432), and p-CREB (CST; #9198) overnight at 4 °C.

Techniques: Expressing, Binding Assay, Membrane, Activation Assay