Journal: Frontiers in Pharmacology

Article Title: Baicalein Attenuates Pyroptosis and Endoplasmic Reticulum Stress Following Spinal Cord Ischemia-Reperfusion Injury via Autophagy Enhancement

doi: 10.3389/fphar.2020.01076

Figure Lengend Snippet: Baicalein enhances autophagy after spinal cord ischemia-reperfusion injury (SCIR). (A) Immunofluorescence staining for LC3II and NeuN colocalization at the spinal cord lesion after SCIR (Scan bar = 25μm). (B) Immunofluorescence staining for p62 and NeuN colocalization at the spinal cord lesion after SCIR (Scan bar = 25μm). (C) The quantitative percentage of the LC3II positive neurons at the spinal cord lesion in each group. (D) The quantitative percentage of the p62 positive neurons in each group. (E) Western blotting for Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II, and p62 expression levels in the Sham, SCIR+Vehicle, and SCIR+Baicalein groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II, and p62 expression levels were quantified and analyzed in each group. The values are expressed as the means ± SEM, n=5 per group. * p < 0.05 and ** p < 0.01, vs. Sham group. ## p < 0.01, vs. SCIR+Vehicle group.

Article Snippet: The rabbit monoclonal anti-phosphoinositide-3-kinase (VPS34) and anti-cathepsin D (CTSD) antibodies were acquired from the Proteintech Group (12452-1 and 21327-1; Chicago, IL, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Baicalein Attenuates Pyroptosis and Endoplasmic Reticulum Stress Following Spinal Cord Ischemia-Reperfusion Injury via Autophagy Enhancement

doi: 10.3389/fphar.2020.01076

Figure Lengend Snippet: Inhibition of autophagy reverses the effects of baicalein on endoplasmic reticulum (ER) stress, pyroptosis, and functional recovery after SCIR. (A) Western blotting for the Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II and p62 expression levels in the SCIR+Baicalein, and SCIR+3MA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (B) The optical density values of the Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II and p62 expression levels were quantified and analyzed in each group. (C, D) Western blotting for the NLRP3, GSDMD, C-CASP1, GRP78, PDI, ATF4, CHOP, and CASP12 expression levels in the SCIR+Baicalein and SCIR+3MA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (E, F) The optical density values of GRP78, PDI, ATF4, CHOP, CASP12, NLRP3, GSDM, and C-CASP1expression levels were quantified and analyzed in the both groups. (G, H) Enzyme-linked immunosorbent assay (ELISA) results for IL1βand IL18 expression levels in the SCIR+Baicalein and SCIR+3MA groups. (I, J) The BMS scores of the mice in each group at 24 h and 7 days after SCIR. The values are expressed as the means ± SEM, n=5 per group. * p < 0.05 and ** p < 0.01, vs. SCIR+Baicalein group.

Article Snippet: The rabbit monoclonal anti-phosphoinositide-3-kinase (VPS34) and anti-cathepsin D (CTSD) antibodies were acquired from the Proteintech Group (12452-1 and 21327-1; Chicago, IL, USA).

Techniques: Inhibition, Functional Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: Alternative polyadenylation upon CPSF6 knock-out enhances HIV-1 infection in primary T cells

doi: 10.1371/journal.ppat.1013745

Figure Lengend Snippet: 3’ UTRs are designated shortened (blue) for Compositional Fold Change (cFC) < -0.25 and -log 10 (adjusted p-value) < 0.05 and lengthened (red) for cFC > 0.25 and -log 10 (adjusted p-value) < 0.05. Dots represent individual altered 3’ UTRs. b, Tracks show read coverage of TGFBR1 in RNA-Seq data from CPSF6 knock-out and NT control cells from one representative biological replicate (donor E), visualized using Integrative Genomics Visualizer. 3’ UTRs show evidence of shortening in response to CPSF6 knock-out. c, Charts show functional enrichment (Metascape) analysis of top pathways enriched in genes with shortened 3’ UTRs (left, blue) and lengthened 3’ UTRs (right, red) in response to CSPF6 knock-out in primary CD4+ T cells as assessed by REPAC analysis in 3 biological replicates (donors E-G). d, Violin plot shows comparison of cFC values from REPAC analysis in all tested genes as compared to genes in the indicated pathways (GO:0042110:T Cell activation, R-HSA-1280215: Cytokine Signaling in Immune system, R-HSA-913531: Interferon Signaling) in 3 biological replicates (donors E-G). e, Scatterplot shows cFC of all tested APA sites in REPAC analysis of CPSF6 knock-out primary CD4+ T cells in as compared to NT controls plotted against differential expression data as shown in in data aggregated from 3 biological replicates (donors E-G). 3’ UTRs are designated shortened (blue) for cFC < -0.25 and -log 10 (adjusted p-value) < 0.05 and lengthened (red) for cFC > 0.25 and -log 10 (adjusted p-value) < 0.05. Dots represent individual altered 3’ UTRs. f, Immunoblot shows knock-out of CPSF5 in protein lysates harvested at day 4 post-editing in 3 biological replicates (donor Z-AB). g, HIV-1 infectivity (% GFP positive cells normalized to NT control per donor) at day 5 post-challenge with HIV-1 NL4-3 nef:IRES:GFP in indicated knock-out primary CD4+ T cells from 3 biological replicates (donors Z-AB) as assessed by flow cytometry. Each bar represents the average of technical triplicates + /- SD with individual data points shown. Statistics were calculated relative to the NT control per condition by one-way ANOVA with Dunnet’s test for multiple comparisons; * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Primary antibodies used in this study are as follows: CPSF6 (Rabbit, 1:3000 in 5% BSA, Novus, #NBP1–85676), CPSF5 (Rabbit, 1:5000 in 4% milk, Proteintech, #10322–1-AP), CYPA (Rabbit, 1:12000 in 4% milk, Cell Signaling, #2175), IFNAR1 (Rabbit, 1:1000 in 4% milk, abcam, #ab124764 [clone EPR6244]), MX1 (Rabbit, 1:1000 in 5% BSA, Cell Signaling, #37849S [clone D3W7I]), IFIT1 (Rabbit, 1:1000 in 4 milk, Cell Signalling #14769 [clone D2X9Z], TRIM5α (1:1000 in 4% milk, Cell Signaling, #14326 [clone D6Z8L]), and β-actin (Mouse, 1:10000 in 4% milk, Cell Signaling, #3700S [clone 8H10D10]).

Techniques: RNA Sequencing, Knock-Out, Control, Functional Assay, Comparison, Activation Assay, Quantitative Proteomics, Western Blot, Infection, Flow Cytometry

Journal: Oncogene

Article Title: Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

doi: 10.1038/onc.2010.585

Figure Lengend Snippet: Figure 1 Effect of T3 on the amount of cysteine proteases and CTSH mRNA and protein in HepG2-TR cell lines. HepG2 cell lines that consistently express wild-type TRa1 (HepG2-TRa1#1, #2) and TRb1 (HepG2-TRb1) were used. As a control, HepG2 cells were transfected with the empty vector, yielding a cell line expressing the Neo protein (HepG2-Neo cells). (a) The CTSH mRNA expression level in four TR stable lines. HepG2-TRa1 or b1 cell lines were incubated for 24–72 h in the absence or presence of T3, after which total RNA was isolated and subjected (20 mg per lane) to Northern blot analysis with 32P-labeled CTSH and 18S RNA probes. Positions of the 1.5-kb CTSH and 18S RNAs are indicated. Student’s t-test. **Po0.01; *Po0.05, T3-depleted (Td) vs 1 or 10 nM T3 treated. (b) Three TR stable lines and Neo cells were incubated in Td medium in the absence or presence of T3 for 24–72 h, after which cell lysates (20 mg protein) were subjected to immunoblot analysis with antibodies to CTSH. The pro-form (upper arrow) and active form (lower arrow) of CTSH band is shown. Actin was used as an internal control. (c) Quantitative real-time PCR analysis of CTSH mRNA in Huh7 cells after 1–10 nM T3 for 24–48 h treatment. (d) Western blot analysis of total cell lysates or extracellular forms of CTSH in Huh7 cells. Huh7 cells were incubated in serum-free medium and 1–10 nM T3 for 24 and 48 h on 10 cm dishes. The culture medium was collected and 20 mg of media were subjected to immunoblot analysis with CTSH antibodies and loading control by using coomassie blue stained. Data are means±s.e. of values from three independent experiments.

Article Snippet: Formalin-fixed and paraffin-embedded tissues from the livers or lungs of SCID mice were examined by immunohistochemistry using a polyclonal antibody to CTSH (Proteintech Group, Inc., Chicago, IL, USA) following the avidin–biotin complex method as described previously (Chen et al., 2008a).

Techniques: Control, Transfection, Plasmid Preparation, Expressing, Incubation, Isolation, Northern Blot, Labeling, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Oncogene

Article Title: Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

doi: 10.1038/onc.2010.585

Figure Lengend Snippet: Figure 2 Regulation of CTSH expression by T3 at the transcriptional level. (a) Schematic representation of CTSH promoter with potential TRE-binding sites indicated by oval shapes. The numbers beside each construct indicate the base pair at which the site starts based on the transcription start site ( þ 1). A series of pA3TK-luc reporter constructs containing progressively smaller amounts of CTSH 50-flanking DNA (1 mg per well) were co-transfected with pcDNA-TRa1#1 in HepG2 cells. After transfection, cells were harvested after T3 treatment for 24 h. Data were normalized to co-transfected b-galactosidase, and fold-activation was calculated relative to each pA3TK-luc control. Results are presented as means s.d. of data from three independent experiments performed in triplicate. (b) In vitro-translated TRa1 or TRb1 incubated with wild-type or mutated TRE of CTSH gene promoter 2038 to 1966 along with TNT in vitro-translated RXRa in a final volume of 20 ml. Antibody C4 is a mouse monoclonal antibody against TR proteins, and MOPC21 is a nonspecific monoclonal antibody. The position of probe complexed with TR and RXR is indicated by the arrows. SS: TR complexes supershifted by C4 or RXRa antibody. The wild-type or mutated TRE of CTSH gene promoter 1565 to 1501 were amplified and labeled with [a-32P-dCTP] as probe.

Article Snippet: Formalin-fixed and paraffin-embedded tissues from the livers or lungs of SCID mice were examined by immunohistochemistry using a polyclonal antibody to CTSH (Proteintech Group, Inc., Chicago, IL, USA) following the avidin–biotin complex method as described previously (Chen et al., 2008a).

Techniques: Expressing, Binding Assay, Construct, Transfection, Activation Assay, Control, In Vitro, Incubation, Labeling

Journal: Oncogene

Article Title: Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

doi: 10.1038/onc.2010.585

Figure Lengend Snippet: Figure 3 Identification of the TR proteins that are associated with the TRE regions of CTSH gene promoter in vivo. (a) Nuclear extracts (NE) were isolated from HepG2-TRa#1 cells, and 5 mg of protein were incubated with [a-32P-dCTP] labeled probes men- tioned in Figure 2. A upstream region 2264 to 2054 in the CTSH promoter gene devoid of the TRE region was a nonspecific (ns) probe. (b) ChIP assay as described in Materials and methods section. DNA corresponding to TRE of CTSH promoter region was analyzed by PCR with the indicated primers. Furin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter region was used as a positive or negative control, respectively. Another positive control was used chromatin before immunopre- cipitation (input) and negative controls were immunoprecipitates with MOPC21 antibody. ChIP assays were also performed in HepG2-TRa or b cells.

Article Snippet: Formalin-fixed and paraffin-embedded tissues from the livers or lungs of SCID mice were examined by immunohistochemistry using a polyclonal antibody to CTSH (Proteintech Group, Inc., Chicago, IL, USA) following the avidin–biotin complex method as described previously (Chen et al., 2008a).

Techniques: In Vivo, Isolation, Incubation, Labeling, Negative Control, Positive Control

Journal: Oncogene

Article Title: Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

doi: 10.1038/onc.2010.585

Figure Lengend Snippet: Figure 4 The CTSH was regulated by TR in thyroidectomized rats and human HCC specimens. (a) Induction of CTSH expression by thyroid hormone in rat liver. The protein expression of CTSH in normal (N, sham-operated), TX, or TX þ T3 male Sprague–Dawley rat liver was determined by western blot analysis. Student’s t-test. **Po0.01, TX þ T3 or N vs TX treated. (b) The mRNA expression of CTSH was determined by quantitative reverse transcription (Q-RT)–PCR. (c) TR and CTSH protein overexpression in 15 representative pairs of human HCC specimens. The band intensities were quantified and normalized to actin. The normalized tumor/ non-tumor (T/N) ratio is shown in the right panel.

Article Snippet: Formalin-fixed and paraffin-embedded tissues from the livers or lungs of SCID mice were examined by immunohistochemistry using a polyclonal antibody to CTSH (Proteintech Group, Inc., Chicago, IL, USA) following the avidin–biotin complex method as described previously (Chen et al., 2008a).

Techniques: Expressing, Western Blot, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Over Expression

Journal: Oncogene

Article Title: Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

doi: 10.1038/onc.2010.585

Figure Lengend Snippet: Figure 5 Functional assay of CTSH in hepatoma cells. (a) Overexpression of CTSH promotes migration and invasion. In all, 20 mg of media were subjected to immunoblot analysis with CTSH antibodies. Bottom was shown as loading control using coomassie blue stained. Overexpression of CTSH increases (b) migration and (c) invasion. J7 cells stably expresses CTSH (CTSH#1, #2 clones) and J7 cells stably expresses control vector (control) migration over a time period of 24 h was quantified with a Transwell migration assay and an invasion assay through a Matrigel-coated membrane. The mean migration or invasion of J7-vector cells was assigned a value of 1. Student’s t test. **Po0.01; *Po0.05, CTSH#1, #2 or pool vs vector. (d) Two Huh7 CTSH knockdown (KD) stable lines (CTSH- KD#1, #2), one pool (CTSH-KD pool) stable cells and one Huh7 luciferase knockdown control cell line (KD-control) were used. Knockdown of CTSH in Huh7 cells was confirmed by western blot in cell lysate or conditioned medium. Knockdown of CTSH reduces (e) migration and (f) invasion. Cell image was shown in the bottom of b, c, e and f.

Article Snippet: Formalin-fixed and paraffin-embedded tissues from the livers or lungs of SCID mice were examined by immunohistochemistry using a polyclonal antibody to CTSH (Proteintech Group, Inc., Chicago, IL, USA) following the avidin–biotin complex method as described previously (Chen et al., 2008a).

Techniques: Functional Assay, Over Expression, Migration, Western Blot, Control, Staining, Stable Transfection, Clone Assay, Plasmid Preparation, Transwell Migration Assay, Invasion Assay, Membrane, Knockdown, Luciferase

Journal: Oncogene

Article Title: Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

doi: 10.1038/onc.2010.585

Figure Lengend Snippet: Figure 6 Mediated overexpression of CTSH by thyroid hormone promotes hepatocyte migration. (a) J7-control or J7-CSTH SCID mice (n ¼ 4 per group) were established. All analyses were carried out 7 weeks after inoculation of tumor cells. Immunohistochemical staining of metastasized J7-control or J7-CSTH cells in lung sections are displayed. The metastasis index (fold, density of tumor numbers in J7-CSTH or J7-control per cm2 area) in lung is shown in the lower panel. (b) Effect of T3-mediated migration activity in HepG2-TRa#1 and Huh7 cells, which knocked down CTSH expression. HepG2-TRa or Huh7 cells were transient transfected with either control (luc short hairpin RNA (shRNA)) or CTSH shRNA by electroporation. After puromycin selection, stable clones were pooled for migration assay. The cell lines were added to the upper chamber of Transwell units and incubated in the absence or presence of T3 (10 nM) for 24 h. The number of cells that transversed the filter to the lower chamber was then determined and expressed as the relative migration activity. (c) Effect of anti-green fluorescent protein (GFP) or anti-CTSH antibody treatment on migration activity in HepG2-TRa#1 and Huh7 cells. The cell lines were added to the upper chamber of Transwell units and incubated in the absence or presence of T3 (10 nM) and anti-GFP or anti-CTSH antibody (10 mg/ml) for 24 h. (d) Two different control cell clones (J7-control#1 and J7-control#2) that stably express neo proteins and two J7-CTSH stable clones were used for MMP assays. J7-control or J7-CSTH cells (2 106) were plated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. After 24-h incubation and subsequent washing, the medium was replaced with serum-free medium. Medium was collected for MMP detection. Gelatin zymography was performed; the position of proenzyme and active form of MMPs is shown to the left. (e) HepG2-TRa#1 and J7- derived cells were treated in serum-free medium and were incubated in T3 (0, 1 and 10 nM) for 24 or 48 h. Medium was collected for western blot analysis of MMP3. (f) J7-control or J7-CSTH cells were cultured in serum-free medium for 24 h. Cell lysates were western blotted with anti-ERK and anti-phospho-ERK antibodies. Actin was used as an internal control. (g) HepG2-TRa#1 or Huh7 cells were stimulated to migrate T3 by in the presence or absence of indicated MEK inhibitors (U0126). The relative migration means that in response to T3 ( þ or – inhibitor) normalized to dimethylsulphoxide (DMSO) treated, T3-deprived controls. Data are means±s.e. of values from three independent experiments.

Article Snippet: Formalin-fixed and paraffin-embedded tissues from the livers or lungs of SCID mice were examined by immunohistochemistry using a polyclonal antibody to CTSH (Proteintech Group, Inc., Chicago, IL, USA) following the avidin–biotin complex method as described previously (Chen et al., 2008a).

Techniques: Over Expression, Migration, Control, Immunohistochemical staining, Staining, Activity Assay, Expressing, Transfection, shRNA, Electroporation, Selection, Clone Assay, Incubation, Stable Transfection, Zymography, Derivative Assay, Western Blot, Cell Culture