Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Epidermal growth factor induces cytokeratin 19 expression accompanied by increased growth abilities in human hepatocellular carcinoma.

doi: 10.1038/labinvest.2010.161

Figure Lengend Snippet: Figure 3 Alteration of CK19 and AFP expression in PLC-5 after EGF stimulation Immunofluorescence staining of CK19 and AFP was performed for PLC-5 with and without EGF treatment. CK19 and AFP were visualized by the Vector Red alkaline phosphatase substrate (red), and nuclei were stained with 406- diamino-2-phenylindole (blue). EGF markedly induced CK19 expression in PLC-5, and the expression of AFP was reduced. Negative controls for CK19 and AFP immunofluorescence staining were shown in the insets. Original magnifications, x200.

Article Snippet: CK19 and AFP were visualized by the Vector Red alkaline phosphatase substrate (red), and nuclei were stained with 406- diamino-2-phenylindole (blue).

Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 2. CuB induced NLRP3/caspase-1/GSDMD-mediated pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, and HMGB1 in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Immunofluorescence, Fluorescence, Co-Immunoprecipitation Assay, Double Staining

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 3. ROS promoted accumulation of pyroptosis-related Tom20. a JC-1 kit was used to examine the MMP levels in A549 cells (Scale bar=25 µm). b, c ROS level was analyzed by flow cytometry in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). d Effect of NAC on morphological feature changes in A549 cells (Scale bar=25 µm). e Western blotting analysis of the expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1 and cleaved caspase-1 in A549 cells. f Annexin V and 7-AAD double staining assay was used to identify the inhibition of NAC CuB-induced pyroptosis by NAC in A549 cells. g Immunofluorescence detection of the expression of mitochondrial protein Tom20 in A549 cells (Scale bar=10 µm). h The cell viability in Tom 20 knockdown A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs si-Ctrl group).

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Flow Cytometry, Control, Western Blot, Expressing, Double Staining, Inhibition, Immunofluorescence, Knockdown

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 4. Cytosolic calcium accumulation was essential for CuB-induced pyroptosis. a, b Effect of BAPTA-AM (10 μM) or VX765 (60 µm) on Ca2+ levels in A549 cells (n ≥3, **P＜0.01, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). c The process of the increase of Ca2+ levels in A549 cells was detected by using a Bio Tek CYTATION5 (Scale bar=25 µm). d Immunofluorescence assay was used to examine the expression of Ca2+ fluorescence (Scale bar=7.5 µm). e Effect of BAPTA-AM on the LDH release in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs control group). f Annexin V/7-AAD double staining assay was used to identify the inhibition of CuB-induced pyroptosis by BAPTA-AM in A549 cells. g The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1 and cleaved caspase-1 in A549 cells.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Control, Immunofluorescence, Expressing, Fluorescence, Double Staining, Inhibition

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 5. TLR4 activation promoted the CuB-induced pyroptosis. a The protein expression of TLR4 in A549 cells. b The molecular docking data of CuB on TLR4 dimer were analyzed by using autodock software. c CETSA-WB experiment to further confirm that CuB targeted TLR4 proteins. d The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, pro-caspase-1, and cleaved caspase-1 in A549 cells. e Annexin V/7-AAD double staining assay was used to identify the inhi bition of CuB-induced pyroptosis by TAK242 in A549 cells. f The protein expression of TLR4 in A549 cells. g, h The cell viability and LDH activity in TLR4 knockdown A549 cells (n ≥3, *** P＜0.001 vs CuB; ###P＜0.001 vs NC group). i, j Mitochondrial ROS and cytosolic Ca2+ levels were detected by flow cytometry in A549 cells (n ≥3, ***P＜0.001 vs CuB; ###P＜0.001 vs NC group).

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Software, Double Staining, Activity Assay, Knockdown, Flow Cytometry

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 7. CuB induced lung cancer cells pyroptosis in mouse models. a HE staining of heart, liver, spleen, lung, and kidney tissues in indicated groups (HE, original magnification, 200×, Scale bar=50 µm). b, c The mitochondrial ROS and cytosolic Ca2+ levels in tumor cells were detected by flow cytometry (n ≥3, ***P＜0.001 vs model group, ###P＜0.001 vs CuB 0.75 mg/kg group). d, e The protein expressions of TLR4, NEK7, NLRP3, ASC, Tom20, GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL- 1β, pro-caspase-1, cleaved caspase-1and HMGB1 in tumor tissues were detected by western blotting.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: World Journal of Gastroenterology : WJG

Article Title: Deletion of Gpr128 results in weight loss and increased intestinal contraction frequency

doi: 10.3748/wjg.v20.i2.498

Figure Lengend Snippet: Targeted deletion of Gpr128 in mice. A: Gene targeting strategy. Numbered boxes represent Gpr128 coding exons. The start codon and stop codon are indicated as a star and pound sign, respectively. The targeting vector contains a 7.1-kb 5’ arm and a 5.3-kb 3’ arm. Exons 10, 11 and 12 of the Gpr128 gene were replaced by a PGK-Neo cassette through homologous recombination. The primer pairs for polymerase chain reaction (PCR) genotyping are indicated by arrows (5’ arm: P1, P2; 3’ arm: P3, P4); B: PCR screening for targeted embryonic stem (ES) cell clones. Correctly recombined clones show 7.7 and 5.7 kb bands, respectively. Three recombined ES cell clones show the expected bands as detected with primers P1-P4; C: PCR analysis of genomic tail DNA derived from Gpr128+/- mouse intercrossing. A 5.4-kb fragment amplified with primers P5 and P6 represents the wild-type (WT) allele. A 5.7-kb band was amplified from the targeted allele with P3 and P4; D: Gpr128 expression in gastrointestinal tissue with two different genotypes by semiquantitive reverse transcription-polymerase chain reaction. A specific Gpr128 fragment, which exists in WT mice, was deleted in Gpr128-/- mice. The transcript for β-actin was examined as a control for RNA loading and integrity; E: Expression pattern of Gpr128 protein in WT and Gpr128-/- adult mouse colon revealed by immunofluorescence (original magnification, × 200). M: Marker lane; (-): Negative control without template; S. intestine: Small intestine; P. colon: Proximal colon; D. colon: Distal colon.

Article Snippet: A 1-μg sample of total RNA was reverse-transcribed to cDNA with an RNA PCR kit (Takara, Dalian, Liaoning, China) according to the standard protocol.

Techniques: Plasmid Preparation, Homologous Recombination, Polymerase Chain Reaction, Clone Assay, Derivative Assay, Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Marker, Negative Control

Journal: World Journal of Gastroenterology : WJG

Article Title: Deletion of Gpr128 results in weight loss and increased intestinal contraction frequency

doi: 10.3748/wjg.v20.i2.498

Figure Lengend Snippet: Selective expression of Gpr128 within the intestine in mice. A: Expression levels of Gpr128 mRNA. The mRNA levels were examined in major tissues of normal mice using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression level of β-actin was used as an endogenous control. M: Marker lane; (-): Negative control without template; B: Northern blotting analysis of Gpr128. Total RNA from wild type mice was extracted and subjected to Northern blotting analysis using a 715-bp fragment of Gpr128 cDNA corresponding to exons 1 through 6. The bottom lane shows the 28S and 18S ribosomal RNA as a control; C: Examination of the stage-specific expression of Gpr128 mRNA. RT-PCR was performed throughout the digestive tract and at various postnatal developmental stages to determine the presence of Gpr128 mRNA from postnatal day 0 through 8 wk; D: Expression pattern of Gpr128 protein in the stomach and colon of adult WT mouse revealed by immunofluorescence (original magnification, × 200).

Article Snippet: A 1-μg sample of total RNA was reverse-transcribed to cDNA with an RNA PCR kit (Takara, Dalian, Liaoning, China) according to the standard protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control, Northern Blot, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Long non-coding RNA LINC00672 contributes to p53 protein-mediated gene suppression and promotes endometrial cancer chemosensitivity

doi: 10.1074/jbc.M116.758508

Figure Lengend Snippet: LINC00672 contributes to the p53-dependent LASP1 suppression. A, association between log2-transformed LINC00672 and LASP1 expression in a set of randomly selected 62 EC tumor and paired adjacent non-neoplastic tissues. B, LINC00672 and LASP1 expression levels detected by qPCR in EC cells after transfection with LINC00672, LINC00672-mutant, and control lentiviruses. Data shown are the mean ± S.D. of three independent experiments, normalized to GAPDH (*, p < 0.05). C, LINC00672 and LASP1 expression levels detected by qPCR in EC cells after transfection with LINC00672 LNA#1, LINC00672 LNA#2 and LNA scramble. Data shown are the mean ± S.D. of three independent experiments, normalized to GAPDH (*, p < 0.05). D, immunofluorescence analysis of LASP1 in EC cells after transfection with LINC00672, LINC00672-mutant, and control lentiviruses. Right panel is the quantification of relative fluorescence mean density (n = 6, *, p < 0.05). E, immunofluorescence analysis of LASP1 in EC cells after transfection with LINC00672 LNA#1, LINC00672 LNA#2, and LNA scramble. Right panel is the quantification of relative fluorescence mean density (n = 6, *, p < 0.05). F, LASP1 levels in HEC-1A cells detected by Western blotting after transfection with LINC00672 lentiviruses, LINC00672-mutant lentiviruses, control lentiviruses, LINC00672 LNA#1, LINC00672 LNA#2, and LNA scramble. G, schema of RNA pulldown experiment followed by Western blotting for verification of hnRNP F/H association (upper). Western blotting results of hnRNP F, H, D, and I in the protein extractions of LINC00672-pulldown are shown. H and I, RIP experiments were performed using hnRNP F, H, D, and I and p53 antibody to immunoprecipitate and a primer to detect LINC00672 in HEC-1A and Ishikawa cells.

Article Snippet: An immunofluorescence analysis of LASP1 was performed using an immunofluorescence staining kit (Beyotime Institute of Biotechnology, China) following the manufacturer's instructions.

Techniques: Transformation Assay, Expressing, Transfection, Mutagenesis, Control, Immunofluorescence, Fluorescence, Western Blot