Journal: Oxidative Medicine and Cellular Longevity

Article Title: Targeting the Ang2/Tie2 Axis with Tanshinone IIA Elicits Vascular Normalization in Ischemic Injury and Colon Cancer

doi: 10.1155/2021/7037786

Figure Lengend Snippet: Tan IIA improved blood perfusion recovery in the ischemic hind limbs. (a) Schematic diagram for depicting the establishment of the combined mouse model of HT-29 xenograft and hind limb ischemia, including the schedule of Tan IIA treatments. (b) Representative images of LDPI of the ischemic hind limbs in mice treated with 0.3% CMC-Na or Tan IIA at the indicated time points. (c) Hindlimb blood flow expressed as a percentage of ischemic limb blood flow over nonischemic hindlimb blood flow measured at the indicated time points ( n = 6). (d) Morphology assessment of ischemic hind limbs in mice treated with 0.3% CMC-Na or Tan IIA at the indicated time points ( n = 8). (e) Representative images of H&E staining for the gastrocnemius muscle at 21 days postsurgery. Scale bar, 50 μ m. (f) Histological scoring of H&E staining for the mice treated with 0.3% CMC-Na or Tan IIA ( n = 8). (g) Hypoxia in the gastrocnemius muscle tissues at day 21 postsurgery was measured by CA-9 staining (brown). Representative images are shown. Scale bar, 50 μ m. (h) Statistical analysis of CA-9 expression in the gastrocnemius muscle tissues ( n = 3). (i) Representative immunofluorescence images of Dystrophin (green) to reflect functional muscle fibers in the gastrocnemius muscle tissues are shown. Scale bar, 25 μ m. The data were presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (versus model group).

Article Snippet: The immunohistochemistry assay for hypoxia was performed using CA-9 (11071-1-AP, Proteintech) antibody as previously described.

Techniques: Staining, Expressing, Immunofluorescence, Functional Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Targeting the Ang2/Tie2 Axis with Tanshinone IIA Elicits Vascular Normalization in Ischemic Injury and Colon Cancer

doi: 10.1155/2021/7037786

Figure Lengend Snippet: Tan IIA induced normalization of tumor blood vessels. (a) Growth curve of HT-29 tumors in the mice treated with 0.3% CMC-Na and Tan IIA (10 mg/kg, 30 mg/kg, and 90 mg/kg) ( n = 8). (b) Representative picture of HT-29 tumors harvested from mice treated with 0.3% CMC-Na and Tan IIA at day 21 postsurgery ( n = 8). (c) Tumor weights from different groups of mice were measured on day 21 postsurgery ( n = 8). (d) Representative immunofluorescence images of PDGFR β and collagen IV in the tumor blood vessels are shown. Scale bars, 25 μ m. (e) Quantification of PDGFR β and collagen IV expression in the tumor blood vessels ( n = 3). (f) Representative images of tumor vascular leakiness in the mice treated with 0.3% CMC-Na and Tan IIA. TRITC-dextran was intravenously injected into BALB/c nude mice bearing HT-29 tumors. The extravasated TRITC-dextran from tumor blood vessels stained for CD31 is shown. (g) The TRITC-dextran leakage was quantified by the ratios of dextran + area to CD31 + area ( n = 3). Scale bars, 50 μ m. (h) Representative immunofluorescence images of Claudin 5 in the tumor blood vessels are shown. Scale bars, 25 μ m. (i) Quantification of Claudin 5 expression in the tumor blood vessels ( n = 3). (j) Hypoxia in the tumor parenchyma was determined by CA-9 staining (brown) at day 21 postsurgery. Representative immunohistochemical staining images are shown. Scale bar, 25 μ m. (k) Quantification of CA-9 expression in the HT-29 tumors harvested from the mice treated with 0.3% CMC-Na and Tan IIA ( n = 3). The data were presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (versus model group).

Article Snippet: The immunohistochemistry assay for hypoxia was performed using CA-9 (11071-1-AP, Proteintech) antibody as previously described.

Techniques: Immunofluorescence, Expressing, Injection, Staining, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma

doi: 10.1038/s41419-022-05171-3

Figure Lengend Snippet: A – C Tumor growth ( A ) and tumor weights ( B ) were measured in CRC and NPC xenografts receiving vehicle or anti-VEGF treatment. The tumor inhibition ratios were calculated ( C ) ( n = 6 mice per group). D Representative micrographs of Ki67 + proliferative cells and cleaved caspase-3 + apoptotic cells in vehicle- or anti-VEGF-treated CRC and NPC tumors. Scale bar = 50 μm. Quantification of Ki67 + , cleaved caspase-3 + signals, and proliferation/apoptosis index (PA index) in vehicle- or anti-VEGF-treated CRC and NPC cancers ( n = 8 random fields per group). E Representative micrographs of CD31 + microvessels and CA9 + hypoxic areas in vehicle- or anti-VEGF-treated CRC and NPC tumors. Scale bar in upper panel = 100 μm, scale bar in lower panel = 50 μm. Quantification of CD31 + tumor vessel parameters and CA9 + signals in vehicle- or anti-VEGF-treated CRC and NPC tumors ( n = 8 random fields per group). F – H Tumor growth ( F ) and tumor weights ( G ) were measured in CRC and NPC xenografts receiving vehicle or sunitinib treatment. The tumor inhibition ratios were calculated ( H ) ( n = 6 mice per group). *** P < 0.001. NS not significant. Data presented as mean ± SD.

Article Snippet: Tissue slides were stained with a rabbit anti-Ki67 antibody (Cat. No. ab16667, Abcam, 1:200); a rabbit anti-Cleaved caspase-3 antibody (Cat. No. 9664, Cell Signaling Technology, 1:100); a rabbit anti-CA9 antibody (Cat. No. 11071-1-AP, Proteintech, 1:200), and a rabbit anti-FGF-2 antibody (Cat. No. A0235, ABclonal, 1:100).

Techniques: Inhibition

Journal: Cell Death & Disease

Article Title: Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma

doi: 10.1038/s41419-022-05171-3

Figure Lengend Snippet: A FGF2 expression levels in SW480 CRC, MDA-MB-231 breast cancer, SK-MEL-5 melanoma, HepG2 hepatocellular carcinoma, A549 lung cancer, PANC-1 pancreatic ductal adenocarcinoma, HONE-1 NPC, CNE-1 NPC, and 5–8F NPC cell lines ( n = 3 samples per group). B Tumor growth of scrambled or FGF2 shRNA-transfected NPC tumor ( n = 8 in NPC-sh Scrambled group and 6 in NPC- FGF2 group). C Representative micrographs of Ki67 + proliferative cells and cleaved caspase-3 + apoptotic cells in NPC. Scale bar = 50 μm. Quantification of Ki67 + , cleaved caspase-3 + signals, and PA index in NPC ( n = 8 or 9 random fields per group). D Representative micrographs of CD31 + microvessels and CA9 + hypoxic areas in NPC. Scale bar in upper panel = 100 μm, scale bar in lower panel = 50 μm. Quantification of CD31 + tumor vessel parameters and CA9 + signals in NPC ( n = 8 random fields per group). E Blood samples from tumor-bearing mice were FACS analyzed for EGFP + signals. Quantification of EGFP + circulating tumor cells ( n = 5 samples per group). F Quantification of clones after culturing NPC-bearing mice blood samples for two weeks ( n = 3 samples per group). G Representative graphs of lungs from NPC-bearing mice. Arrows indicate visible metastatic nodules. Scale bar = 0.5 cm. EGFP + metastatic signals in the lung. Quantification of pulmonary metastasis proportion ( n = 6 mice per group). ** P < 0.01; *** P < 0.001. NS not significant. Data presented as mean ± SD.

Article Snippet: Tissue slides were stained with a rabbit anti-Ki67 antibody (Cat. No. ab16667, Abcam, 1:200); a rabbit anti-Cleaved caspase-3 antibody (Cat. No. 9664, Cell Signaling Technology, 1:100); a rabbit anti-CA9 antibody (Cat. No. 11071-1-AP, Proteintech, 1:200), and a rabbit anti-FGF-2 antibody (Cat. No. A0235, ABclonal, 1:100).

Techniques: Expressing, shRNA, Transfection, Clone Assay

Journal: Cell Death & Disease

Article Title: Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma

doi: 10.1038/s41419-022-05171-3

Figure Lengend Snippet: A – D Tumor growth ( A, B ) and tumor weights ( C ) were measured in sh Scrambled - and sh FGF2 -transfected NPC tumors receiving vehicle or anti-VEGF treatment. The tumor inhibition ratios were calculated ( D ) ( n = 6 mice per group). E Representative micrographs of Ki67 + proliferative cells and cleaved caspase-3 + apoptotic cells in vehicle- or anti-VEGF-treated sh Scrambled - and sh FGF2 -transfected NPC tumors. Scale bar = 50 μm. Quantification of Ki67 + , cleaved caspase-3 + signals, and PA index in vehicle- or anti-VEGF-treated sh Scrambled - and sh FGF2 -transfected NPC tumors ( n = 8 random fields per group). F Representative micrographs of CD31 + microvessels and CA9 + hypoxic areas in vehicle- or anti-VEGF-treated sh Scrambled - and sh FGF2 -transfected NPC tumors. Scale bar in upper panel = 100 μm, scale bar in lower panel = 50 μm. Quantification of CD31 + tumor vessel parameters and CA9 + signals in vehicle- or anti-VEGF-treated sh Scrambled - and sh FGF2 -transfected NPC tumors ( n = 8 random fields per group). G Blood samples from tumor-bearing mice were FACS analyzed for EGFP + signals. Quantification of EGFP + circulating tumor cells ( n = 6 samples per group). H Quantification of clones after culturing blood samples from vehicle- or anti-VEGF-treated sh Scrambled - and sh FGF2 -transfected NPC-bearing mice for two weeks ( n = 6 samples per group). I Representative graphs of lungs from vehicle- or anti-VEGF-treated sh Scrambled - and sh FGF2 -transfected NPC-bearing mice. Arrows indicate visible metastatic nodules. Scale bar = 0.5 cm. EGFP + metastatic signals in the lung. Quantification of pulmonary metastasis proportion ( n = 6 mice per group). ** P < 0.01; *** P < 0.001. NS not significant. Data presented as mean ± SD.

Article Snippet: Tissue slides were stained with a rabbit anti-Ki67 antibody (Cat. No. ab16667, Abcam, 1:200); a rabbit anti-Cleaved caspase-3 antibody (Cat. No. 9664, Cell Signaling Technology, 1:100); a rabbit anti-CA9 antibody (Cat. No. 11071-1-AP, Proteintech, 1:200), and a rabbit anti-FGF-2 antibody (Cat. No. A0235, ABclonal, 1:100).

Techniques: Transfection, Inhibition, Clone Assay

Journal: Cell Death & Disease

Article Title: Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma

doi: 10.1038/s41419-022-05171-3

Figure Lengend Snippet: A – C Tumor growth ( A ) and tumor weights ( B ) were measured in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. The tumor inhibition ratio were calculated ( C ) ( n = 6 mice per group). D Representative micrographs of Ki67 + proliferative cells and cleaved caspase-3 + apoptotic cells in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. Scale bar = 50 μm. Quantification of Ki67 + , cleaved caspase-3 + signals, and PA index in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. ( n = 8 random fields per group) E Representative micrographs of CD31 + microvessels and CA9 + hypoxic areas in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. Scale bar in upper panel=100 μm, scale bar in lower panel=50 μm. Quantification of CD31 + tumor vessel parameters and CA9 + signals in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors ( n = 8 or 6 random fields per group). F Blood samples from tumor-bearing mice were FACS analyzed for EGFP + signals. Quantification of EGFP + circulating tumor cells ( n = 6 samples per group). G Quantification of clones after culturing blood samples from vehicle-, anti-VEGF-, and lenvatinib-treated NPC-bearing mice for two weeks ( n = 6 samples per group). H Representative graphs of lungs from vehicle-, anti-VEGF-, and lenvatinib-treated NPC-bearing mice. Arrows indicate visible metastatic nodules. Scale bar = 0.5 cm. EGFP + metastatic signals in the lung. Quantification of pulmonary metastasis proportion ( n = 6 mice per group). I Vehicle- or FGF-2-treated ECs were treated with or without lenvatinib. ERK phosphorylation in ECs was detected. β-actin marks the loading level in each lane ( n = 3 samples per group). J QPCR quantification of Myc mRNA levels in vehicle- or FGF-2-treated ECs, with or without lenvatinib ( n = 3 samples per group). *** P < 0.001. NS not significant. Data presented as mean ± SD.

Article Snippet: Tissue slides were stained with a rabbit anti-Ki67 antibody (Cat. No. ab16667, Abcam, 1:200); a rabbit anti-Cleaved caspase-3 antibody (Cat. No. 9664, Cell Signaling Technology, 1:100); a rabbit anti-CA9 antibody (Cat. No. 11071-1-AP, Proteintech, 1:200), and a rabbit anti-FGF-2 antibody (Cat. No. A0235, ABclonal, 1:100).

Techniques: Inhibition, Clone Assay

Journal: Cell Death & Disease

Article Title: Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma

doi: 10.1038/s41419-022-05171-3

Figure Lengend Snippet: A Schematic diagram of the establishment of humanized NSG mice. B Representative FACS analysis of human CD45 + cells in mouse peripheral blood. Human CD45 + cell percentage greater than 25% was considered successful in modeling. C – E Tumor growth ( C ), tumor weights ( D ) were measured in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. The tumor inhibition ratio were calculated ( E ) ( n = 3 samples per group). F Representative micrographs of Ki67 + proliferative cells and cleaved caspase-3 + apoptotic cells in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. Scale bar = 50 μm. Quantification of Ki67 + , cleaved caspase-3 + signals, and PA index in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. ( n = 8 random fields per group) G Representative micrographs of CD31 + microvessels and CA9 + hypoxic areas in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. Scale bar in upper panel = 100 μm, scale bar in lower panel = 50 μm. Quantification of CD31 + tumor vessel parameters and CA9 + signals in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors ( n = 8 random fields per group). H Quantification of hCD45 + hCD14 + population, hCD45 + hCD19 + population, hCD45 + hCD3 + population, and hCD45 + hCD56 + population in the NPC TME ( n = 3 samples per group). I Quantification of mCD45 + mCD11b + mF4/80 + population, mCD45 + mB220 + population, mCD45 + mCD3 + population, and mCD45 + mCD49b + population in the NPC TME ( n = 3 samples per group). ** P < 0.01; *** P < 0.001. NS not significant. Data presented as mean ± SD.

Article Snippet: Tissue slides were stained with a rabbit anti-Ki67 antibody (Cat. No. ab16667, Abcam, 1:200); a rabbit anti-Cleaved caspase-3 antibody (Cat. No. 9664, Cell Signaling Technology, 1:100); a rabbit anti-CA9 antibody (Cat. No. 11071-1-AP, Proteintech, 1:200), and a rabbit anti-FGF-2 antibody (Cat. No. A0235, ABclonal, 1:100).

Techniques: Inhibition

Journal: eLife

Article Title: Hypoxia-induced proteasomal degradation of DBC1 by SIAH2 in breast cancer progression

doi: 10.7554/eLife.81247

Figure Lengend Snippet: ( A ) SIAH2 expression level in different human cancers from TCGA data in TIMER. *p<0.05, **p<0.01, ***p<0.001. ( B ) Volcano plot analysis of SIAH2 DBC1 and OTUD5 expression levels in breast cancer. ( C ) Relative expression level of SIAH2 in tumor stage (stages 1, 2, 3, 4, or 5) from BRCA patients. ( D ) Relative expression level of SIAH2 in nodal metastasis status (normal, N0, or N1) from BRCA patients. ( E ) Immunohistochemistry staining analysis of the expression levels of SIAH2 and DBC1 in a series of breast cancer patient tissue microarrays. Scale bars, (left) 200 μm; (right) 100 μm. ( F ) Heatmap of the expression levels of SIAH2 and DBC1 in human normal breast tissues and breast tumor tissues. ( G ) Human breast cancer tissues were stained with DAPI (blue) together with anti-CAIX (green) and anti-DBC1 (red) antibodies. Scale bars, 50 μm. ( H ) Statistical analysis of correlations between DBC1 expression level and clinical T stage. ( I ) Statistical analysis of correlations between DBC1 expression level and Ki67-positive stage.

Article Snippet: The following antibodies were used: CAIX (1:100, Proteintech, 66243-1-Ig); DBC1 (1:1000, CST, 5693); and DAPI (300 nM, Invitrogen, D21490).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Bioengineered

Article Title: A novel hypoxia-driven gene signature that can predict the prognosis of hepatocellular carcinoma

doi: 10.1080/21655979.2022.2073943

Figure Lengend Snippet: Exploration of the biological functions of CFHR3 in HCC cells in vitro . (a) Immunohistochemical staining demonstrates that CFHR3 expression is decreased in HCC tissues with high expression levels of hypoxia inducible factor 1 subunit alpha (HIF1A) and carbonic anhydrase 9 (CA9). (b-c) Western blot was used to detect the expression of CFHR3 in HCC cells in the blank control group (BC, without any treatment; cultured under normoxia and hypoxia), in the negative control group (NC, transfected with vector; cultured under normoxia and hypoxia) and in the CFHR3 overexpressing group (CFHR3-OE; cultured under hypoxia). (d-e) 5-ethynyl-2’-deoxyuridine (EdU) assays were used to detect the effects of CFHR3 on HCC cell proliferation under hypoxia. (f-g) Colony formation assays were used to detect the effects of CFHR3 on HCC cell colony formation under hypoxia. (h-i) Transwell assays were used to detect the effects of CFHR3 on HCC cell invasiveness under hypoxia. ** P < 0.01.

Article Snippet: Primary antibodies against HIF1α (1:100; Cat No. 20960-1-AP; Proteintech, Wuhan, China), carbonic anhydrase 9 (CA9; 1:200; Cat No. 11071-1-AP; Proteintech, Wuhan, China), CFHR3 (1:100; Cat No. 16583-1-AP; Proteintech, Wuhan, China), and proliferating cell nuclear antigen (PCNA; 1:200; Cat No. 10205-2-AP; Proteintech, Wuhan, China) were incubated overnight at 4°C.

Techniques: In Vitro, Immunohistochemical staining, Staining, Expressing, Western Blot, Cell Culture, Negative Control, Transfection, Plasmid Preparation

Journal: Bioengineered

Article Title: A novel hypoxia-driven gene signature that can predict the prognosis of hepatocellular carcinoma

doi: 10.1080/21655979.2022.2073943

Figure Lengend Snippet: Exploration of the biological functions of CFHR3 in HCC cells in vivo . (a, b) The proliferation of HepG2 cells with CFHR3 overexpression and NC cells in vivo . (c) Expression levels of HIF1α, CA9, CFHR3, and proliferating cell nuclear antigen (PCNA) in tumor tissues derived from HepG2 cells with CFHR3 overexpression and NC cells. (d, e) Metastatic foci in the lungs from mice injected with SMMC-7721 cells with CFHR3 overexpression and NC cells. ** P < 0.01.

Article Snippet: Primary antibodies against HIF1α (1:100; Cat No. 20960-1-AP; Proteintech, Wuhan, China), carbonic anhydrase 9 (CA9; 1:200; Cat No. 11071-1-AP; Proteintech, Wuhan, China), CFHR3 (1:100; Cat No. 16583-1-AP; Proteintech, Wuhan, China), and proliferating cell nuclear antigen (PCNA; 1:200; Cat No. 10205-2-AP; Proteintech, Wuhan, China) were incubated overnight at 4°C.

Techniques: In Vivo, Over Expression, Expressing, Derivative Assay, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Stimulates SUMOylation-Dependent Stabilization of KDM5B

doi: 10.3389/fcell.2021.741736

Figure Lengend Snippet: Targeting KDM5B to overcome hypoxia adaption in vivo . (A–D) MFC cells were subcutaneously injected into the flanks of nude mice to generate xenograft tumors ( n = 6 ). And they were treated as indicated to evaluate the effect of JIB04, Endostar, and 2-D08 on tumorigenicity. The representative image (A) , tumor growth curve (B) , the tumor weight of xenograft at sacrifice (C) , and the body weight of mice during euthanasia (D) after drug injection for 11 days. (E) IHC staining with CD31 was applied to the section of the xenograft tumors from the control group and Endostar to evaluate the MVD. The presented images were shown (left panel) , and the brown areas represent vascular signals. And the MVD were compared in the two groups (right panel) . (F and G) IHC staining with CA9 (F) and HIF1α (G) was performed to reveal the hypoxia in the control and Endostar groups. (H) A schematic illustration of the model depicting the PIAS4-mediated SUMOylation protected KDM5B from ubiquitination-dependent proteasomal degradation of KDM5B to promote hypoxia adaption of gastric cancer.

Article Snippet: The antibodies used were listed as follows: anti-KDM5B (Abcam ab181089, for WB), anti-KDM5B (Abcam ab211366, for IHC), anti–β-actin (Abclonal ac026), anti-PIAS4 (CST 4392s), anti-FLAG-tag (Sigma-Aldrich F1804-1), anti–HA-tag (earthox, E022010), anti–V5-tag (Invitrogen 46-0705), anti–p-RB (CST 8516S), anti-Rb (santa cruz sc-102), anti-p21 (CST 2946S), anti-cyclin E (CST 20808), anti-UB (Santa cruz sc-8071), anti-His (Proteintech 66,005-1-ig), anti-CD31 (Abcam ab24590), anti-CA9 (Proteintech 11071-1-AP), anti-HIF1α (CST 36169S, for WB), and anti-HIF1α (NB100-105, for IHC).

Techniques: In Vivo, Injection, Immunohistochemistry