Journal: The FEBS journal

Article Title: MAPKs and Hsc70 are critical to the protective effect of molecular hydrogen during the early phase of acute pancreatitis.

doi: 10.1111/febs.13629

Figure Lengend Snippet: Fig. 2. Signaling networks associated with the differentially expressed proteins. To identify the signal transduction pathways and/or signaling networks associated with the differentially expressed proteins, the 73 identified proteins were imported into the MINT, HPRD, and GRID databases. Yellow spots represent the target proteins with a known regulatory relationship. Red spots represent the interacting proteins. (A) Twenty-nine target proteins of the total 73 proteins were used to constructing the protein interaction network shown. (B) MAP4K1, MAP4K5, MAPK1, MAPK3, and Hsc70 (HSPA8) were present in the protein interaction network. (C) Flow chart describing the relationships between members of the MAPK pathway.

Article Snippet: The section were then blocked for 30 min at room temperature and incubated overnight with monoclonal antibodies against p-ERK1/2, p-JNK1/2, p-p38 (1 : 300 dilution, Cell Signaling Technology), or Hsc70 (1 : 400 dilution, Proteintech Group) at 4 °C.

Techniques: Transduction

Journal: The FEBS journal

Article Title: MAPKs and Hsc70 are critical to the protective effect of molecular hydrogen during the early phase of acute pancreatitis.

doi: 10.1111/febs.13629

Figure Lengend Snippet: Fig. 6. Hydrogen upregulated Hsc70 in AP cells. (A) AR42J cells were divided into control, AP, AP+H2, and control+H2 groups. Western blot analysis demonstrated that H2 upregulated the expression of Hsc70 when the AP cells were exposed to H2. The data are expressed as means SD; *P < 0.05. (B) AR42J cells were divided into control, AP, and AP+H2 groups. The effect of H2 on the mRNA level of Hsc70 in pancreatitis cells was further analyzed by real-time PCR. The outcomes were consistent with the protein level results. The data are expressed as means SD; *P < 0.05 compared with the control group, #P < 0.05 compared with the AP group. (C) AR42J cell groups were divided into the normal cells, cells with transfection reagent and cells transfected with control siRNA or Hsc70-specific siRNA, and all four groups were exposed to cerulein and H2. Western blot analysis showed that the levels of NF-jB p65, TNF-a, and IL-1b were all significantly elevated by silencing Hsc70 expression in the AP cells using Hsc70-specific siRNA. No effect was observed in the cells transfected with the control siRNA or exposed to the transfection reagent. The data are expressed as means SD; *P < 0.05. (D) EMSA analysis confirmed that the DNA-binding activity of NF-jB p65 was significantly increased by silencing Hsc70 expression. No effect was observed in the cells transfected with the control siRNA or exposed to the transfection reagent. The data are expressed as means SD; *P < 0.05.

Article Snippet: The section were then blocked for 30 min at room temperature and incubated overnight with monoclonal antibodies against p-ERK1/2, p-JNK1/2, p-p38 (1 : 300 dilution, Cell Signaling Technology), or Hsc70 (1 : 400 dilution, Proteintech Group) at 4 °C.

Techniques: Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Binding Assay, Activity Assay

Journal: The FEBS journal

Article Title: MAPKs and Hsc70 are critical to the protective effect of molecular hydrogen during the early phase of acute pancreatitis.

doi: 10.1111/febs.13629

Figure Lengend Snippet: Fig. 7. Verification experiments in vivo. (A) All animals were randomly allocated into three experimental groups: the sham group was provided normal room air through an anesthetic gas vaporizer utilizing a facial mask after the sham operation; the AP group received the same treatment after inducing AP with a retrograde infusion of 3.5% sodium taurocholate into the pancreatobiliary duct; and the AP+H2 group was administered 2% hydrogen gas supplemented with 98% balanced air through an anesthetic gas vaporizer utilizing a facial mask after inducing AP. All animals were sacrificed 24 h after inducing AP. Western blot analysis verified the outcomes of the in vitro experiments in terms of the p-ERK, p-JNK, p-p38, and Hsc70 levels associated with H2 treatment. H2 inhalation inhibited the activation of the MAPK pathways and increased the protein level of Hsc70. The data are expressed as means SD; *P < 0.05. (B) Histological examination was performed to evaluate the therapeutic effect of H2 in vivo. Immunohistological staining confirmed the results of the previous experiments. The expression of p-ERK, p-JNK, and p-p38 in pancreatic acinar cells was significantly decreased and the Hsc70 protein level was significantly increased after H2 exposure. The scale bar represents 50 lm. (C) Animals were randomly allocated into four experimental groups: the AP+H2 group was treated as described above, and the AP+H2 + PD98059, SP600125 or SB203580 group received an intraperitoneal injection of 1 mL of the corresponding specific inhibitor solution (15 mgkg1) at 30 min before the surgical procedure. All animals were sacrificed 24 h after AP induction. The levels of p-ERK1/2, p-JNK1/2, and p-p38 were all elevated in the AP+H2 group compared with the MAPK-inhibition groups. Combined treatment with H2 and the corresponding specific inhibitor significantly attenuated the increased expression of these proteins. Simultaneously, the levels of these indexes in the other two groups were between those observed in the AP+H2 group, and the AP+H2 + the corresponding specific inhibitor group. The data are expressed as means SD; *P < 0.05.

Article Snippet: The section were then blocked for 30 min at room temperature and incubated overnight with monoclonal antibodies against p-ERK1/2, p-JNK1/2, p-p38 (1 : 300 dilution, Cell Signaling Technology), or Hsc70 (1 : 400 dilution, Proteintech Group) at 4 °C.

Techniques: In Vivo, Western Blot, In Vitro, Activation Assay, Staining, Expressing, Injection, Inhibition

Journal: Frontiers in Microbiology

Article Title: Identification of a Novel Serum Biomarker for Tuberculosis Infection in Chinese HIV Patients by iTRAQ-Based Quantitative Proteomics

doi: 10.3389/fmicb.2018.00330

Figure Lengend Snippet: Differentially expressed proteins in the sera of HIV positive patients with active tuberculosis (TB) relative to controls (only HIV positive patients).

Article Snippet: The concentrations of ENG (CUSABIO, CSB-E10030h, China), PSMB2 (CUSABIO, CSB-E17836h, China), HSP90AA1 (CUSABIO, CSB-E13462h, China), HSPA8 (CUSABIO, CSB-EL010829HU, China), CHI3L1 (CUSABIO, CSB-E13608h, China), CAP1 (CUSABIO, CSB-EL004486HU, China), ORM1 (CUSABIO, CSB-EL017237HU, China), RETN (CUSABIO, CSB-E06884h, China), LPA (CUSABIO, CSB-EQ028005HU, China) and ZYX (CUSABIO, CSB-EL027165HU, China) in the serum samples were quantified using ELISA according to the manufacturer’s instructions.

Techniques:

Journal: Cells

Article Title: D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation

doi: 10.3390/cells11071230

Figure Lengend Snippet: Effect of Na 2 S on the amounts of Nrf2 and CMA-related proteins in AD293 cells. ( A ) Immunoblot analyses of Nrf2 and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 4 and 24 h. ( B ) Quantitative analyses of Nrf2 amounts from the immunoblot results shown in A. ( C ) Immunoblot analyses of CMA-related proteins (LAMP2A, Hsc70) and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 24 h. ( D ) Quantitative analyses of LAMP2A and Hsc70 amounts from the immunoblot results shown in C. ( E ) Immunoblot analyses of a CMA/mA substrate (MEF2D) and β-actin in cell lysates from AD293 cells treated with Na 2 S (10 μM) for 24 h. ( F ) Quantitative analysis of MEF2D amount from the immunoblot results shown in F. Whole blot images are presented in . Amounts of β-actin were used as internal controls for quantification. Numbers in the columns represent the number of samples. * p < 0.05, *** p < 0.001 (unpaired t -test).

Article Snippet: Anti-Hsc70 mouse monoclonal IgM antibody (#NB120-2788) was obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Western Blot

Journal: Cells

Article Title: D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation

doi: 10.3390/cells11071230

Figure Lengend Snippet: Effect of long-term treatment with D-cysteine on the amounts of Nrf2- and CMA-related proteins in cerebellar lysates from ICR mice. ( A ) Immunoblot analyses of Nrf2, NQO1, LAMP2A, Hsc70, MEF2D, and β-actin in cerebellar lysates from ICR mice daily treated with saline (Sal) and D-cysteine (100 mg/kg/day) for 10 weeks. Whole blot images are presented in . ( B ) Quantitative analyses of the amounts of Nrf2, NQO1, LAMP2A, Hsc70, and MEF2D shown in A. Amounts of β-actin were used as internal controls for the quantification. * p < 0.05, ** p < 0.01 vs. saline-treated mice (unpaired t -test, n = 5 in both saline- and D-cysteine-treated mice).

Article Snippet: Anti-Hsc70 mouse monoclonal IgM antibody (#NB120-2788) was obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Western Blot, Saline

Journal: iScience

Article Title: Network pharmacology and experimental validation to reveal the target of matrine against PRRSV

doi: 10.1016/j.isci.2023.106371

Figure Lengend Snippet: Key resources table

Article Snippet: Rabbit polyclonal anti-HSPA8 , ABclonal , Cat#A14001; RRID: AB_2313773.

Techniques: Virus, Isolation, Infection, Recombinant, Control, Protein Concentration, Software

Journal: Science Advances

Article Title: A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation

doi: 10.1126/sciadv.aaz2059

Figure Lengend Snippet: ( A ) Two-dimensional visualization of the single immune cell (CD45 + cells) transcriptome in the dermis of healthy donors ( n = 3) and patients with psoriasis ( n = 3). Immune cell compartments are encircled, and feature plots of MIR155HG expression in different subsets are presented. ( B ) Silver staining of P155 interactive protein in the immunoprecipitants pulled down by streptavidin-agarose from human moDCs pretreated with R848 (1 μg/ml) and biotin-Scr/P155 (25 μM). The black box represents target protein. ( C ) Scatterplot of representative data for intensity of proteins detected with MS in human moDCs treated with R848 (1 μg/ml) and Biotin-Scr/P155 (25 μM). The dots represent the intensities (log10-transformed) of all proteins identified in the P155 group ( y axis) and the Scr group ( x axis), and the purple dot represents the protein of interest. ( D ) Immunoblotting verification of the interaction between HSC70 and P155. The black arrowhead indicates the specific band. ( E ) Immunoblotting detection of the P155-specific binding domain in the immunoprecipitants pulled down by streptavidin-agarose from biotin-Scr/P155–pretreated HEK293T cells overexpressing Myc-Tag–labeled HSC70 subdomain plasmids. Anti–Myc-Tag antibody was used and the black box indicates the specific banding. IB, immunoblot; PD, pull-down assay. ( F ) Confocal visualization of PLA signals (red/pink dots) in HEK293T cells overexpressing Myc-Tag–labeled HSC70 or HSC70 subdomain plasmids together with the endogenously expressed P155. Myc-Tag–labeled HSC70 protein served as a positive control, and the red arrows indicate the specific signals. ( G ) ATPase activity of HSC70 in the presence of P155 or Scr ( n = 3). ( H ) GO pathway enrichment analysis of down-regulated differentially expressed genes (DEGs) of RNA-seq data from THP-1–derived DCs treated with R848 (1 μg/ml) and Scr/P155 (25 μM) compared to Scr-treated controls. Dot color represents the value of −log P ; dot size corresponds to the gene counts, and the red words indicate the pathways we are focusing on. Scale bar, 10 μm. Data (B to E and G) are representative of three independent experiments. ** P < 0.01, two-tailed Student’s t test (mean ± SEM). Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: To determine the HSC70 ATPase activity, the recombinant protein of human heat shock 70-kDa protein 8 (HSPA8) transcript variant 1 (OriGene) was prepared in assay buffer at a final concentration of 0.2 μM with P155 or Scr (25 μM) added to the solution.

Techniques: Expressing, Silver Staining, Transformation Assay, Western Blot, Binding Assay, Labeling, Pull Down Assay, Positive Control, Activity Assay, RNA Sequencing, Derivative Assay, Two Tailed Test

Journal: Science Advances

Article Title: A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation

doi: 10.1126/sciadv.aaz2059

Figure Lengend Snippet: ( A ) Silver staining of P155 interactive protein in the immunoprecipitants pulled down by streptavidin-agarose from murine BMDCs pretreated with R848 (1 μg/ml) and biotin-Scr/P155 (25 μM). The black box represents target protein. ( B ) Scatterplot of representative data for intensity of proteins detected with MS in murine BMDCs treated with R848 (1 μg/ml) and Biotin-Scr/P155 (25 μM). The dots represent the intensities (log10-transformed) of all proteins identified in the P155 group ( y axis) and the Scr group ( x axis), and the purple dot represents the protein of interest. ( C ) Immunoblotting verification of the interaction between HSC70 and P155. The black arrowhead indicates the target band. ( D ) Confocal microscopic images of murine BMDCs treated with FITC-OVA with the cells costained with LAMP2A (red); nuclei were stained with DAPI (blue). The white arrows indicate the colocalization of OVA and LAMP2A (yellow), and the white dashed line is the position indicator for the fluorescence colocation analysis. ( E ) Fluorescence intensity analysis of colocalization of FITC-OVA and LAMP2A in murine BMDCs treated with Scr or P155 ( n = 6). ImageJ software was used to analyze the fluorescence colocalization. The white dashed indicator line of statistical analysis centers on the colocation of FITC-OVA and LAMP2A. ( F ) Immunoblotting detection of HSP90 and LAMP2A expression in the immunoprecipitants pulled down by anti-HSC70 from R848- and Scr/P155-pretreated BMDCs, with immunoprecipitants pulled down by rat IgG from R848- and Scr/P155-pretreated BMDCs serving as the negative controls. The black arrows indicate the target protein. IP, immunoprecipitation; IB, immunoblot. The fold (FD) value represents the protein intensity of HSP90 or LAMP2A and is measured with ImageJ software. ( G ) Confocal microscopic images of the distribution of MHC class II (green) in R848- and Scr/P155-pretreated murine BMDCs. Nuclei were stained with DAPI (blue). ( H ) Representative flow cytometry chart (left) and statistics analysis of OT-II T cell proliferation rates (right, indicated by CD4 + CD44 + Violet − cell percentages) in coculture with BMDCs pretreated with soluble OVA ( n = 3 to 4). ( I ) Schematic diagram of MIR155HG encoding P155 function in regulating DC presentation. Scale bars, 10 μm (D) or 25 μm/5 μm (G). All data are representative of three independent experiments. n.s., not significant; ** P < 0.01 and **** P < 0.0001, one-way ANOVA (mean ± SEM). Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: To determine the HSC70 ATPase activity, the recombinant protein of human heat shock 70-kDa protein 8 (HSPA8) transcript variant 1 (OriGene) was prepared in assay buffer at a final concentration of 0.2 μM with P155 or Scr (25 μM) added to the solution.

Techniques: Silver Staining, Transformation Assay, Western Blot, Staining, Fluorescence, Software, Expressing, Immunoprecipitation, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: Verifying the potential binding of HSPA8 with OC. A Coomassie blue staining of HSPA8. (A) total of 3 µg of purified HSPA8 was loaded for SDS-PAGE followed by Coomassie blue staining. (B) Biacore SPR analysis of HSPA8 and OC binding. HSPA8 was immobilized on the CM5 chip and different concentrations of OC passed through the chip for 60 s. The curves indicate the potential binding between OC and HSPA8. (C) The KD value was calculated from Biacore SPR data. (D) ITC assay of HSPA8 and OC was performed at 25°C. The raw ITC data for injecting OC into the sample cell containing HSPA8 protein. The reaction heat of OC and protein binding was expressed as differential power (DP) between the reference and sample cells. (E) Experimental data are expressed with solid dots and fitted to a binding curve by a one-site binding model. (F) Thermal shift assay of HSPA8 and OC. HSPA8 was incubated with different concentrations of OC, and the melting curve was plotted and calculated using fluorescence intensity.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Binding Assay, Staining, Purification, SDS Page, Isothermal Titration Calorimetry, Protein Binding, Thermal Shift Assay, Incubation, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: Verifying the potential binding of HSPA8 with OC_biotin. (A) Biacore SPR analysis of HSPA8 and OC_biotin potential binding. HSPA8 was immobilized on the CM5 chip and different concentrations of OC_biotin were passed through the chip for 60 s. The curves indicated the potential binding between OC_biotin and HSPA8. (B) The KD value was calculated from Biacore SPR data. (C) ITC measurement of HSPA8 and OC_biotin was carried out at 25°C. The raw ITC data for injecting OC_biotin into the sample cell containing HSPA8 protein. The reaction heat of OC_biotin and protein binding were expressed as differential power (DP) between the reference and sample cells. (D) Experimental data are expressed with solid dots and fitted to a binding curve by a one-site binding model.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Binding Assay, Protein Binding

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: Verifying the potential binding between OC_biotin and HSPA8 in cells. (A) OC_biotin or OC induced GFP-LC3 puncta in HeLa cells. HeLa cells with stable expression of GFP-LC3 were treated with 10 µM OC or 10 µM OC_biotin for 6 h, and GFP fluorescence was detected and analyzed using a high content screening system. Scale bar, 50 µm. (B) Statistical analysis of the number of GFP puncta formed per cell. Data are expressed as mean ± standard deviation of three independent experiments compared with control (** p < 0.01, *** p < 0.001 vs. control). (C) Western blot of LC3B, p62, and PARP. HeLa cells were treated with 10 µM OC or 10 µM OC_biotin for 24 h. The cells were lyzed using RIPA buffer and the protein expression level was analyzed by western blot. (D) Statistical analysis on the relative intensity of LC3BII, P62, cleaved PARP vs. GAPDH. GAPDH protein was used as loading control. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). (E) Immunoprecipitation of HSPA8 and OC_biotin. OC_biotin was incubated with avidin beads followed by immunoprecipitation and western blot. Upper lane: pulldown proteins eluted from avidin beads; middle band: proteins from supernatant after immunoprecipitation; lower band: input HSPA8 protein as control.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Binding Assay, Expressing, Fluorescence, High Content Screening, Standard Deviation, Control, Western Blot, Immunoprecipitation, Incubation, Avidin-Biotin Assay

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: OC and OC_biotin inhibit HSPA8 translocation in cells. (A) Colocalization of OC_biotin with HSPA8. A549 cells were treated with 10 µM OC_biotin or DMSO for 1 h. Cells were fixed with 4% paraformaldehyde and stained with streptavidin and HSPA8 antibodies. Cell nuclei were stained by DAPI. Fluorescent images were acquired using a high content screening system. (B) OC or OC_biotin blocked HSPA8 translocation following stimulation at 42°C. A549 cells were pretreated with 20 µM OC, 20 µM OC_biotin, or 20 µM PES for 1 h, then incubated at 37°C or 42°C for 4 h. The cells were then fixed and stained with HSPA8 antibody and DAPI. (C) Statistical analysis on translocation of HSPA8 from the cytosol to the nucleus. A549 cells were treated with different concentrations of OC, OC_biotin, or PES. The cells were fixed and stained with the HSPA8 antibody. The intensity ratio of the nucleus and cytosol was automatically calculated by a high content screening system. Data are expressed as mean ± standard deviation ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle control). Scale bar, 50 µm.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Translocation Assay, Staining, High Content Screening, Incubation, Standard Deviation, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: OC promotes apoptosis upon cisplatin treatment. (A) OC increased p53 expression level when co-treated with cisplatin. A549 cells were treated with the combination of 10 µM OC, 40 µM cisplatin, and 10 µM PES for 24 h. Western blot analysis showed the changes in p53 and LC3B cleavage. (B) Statistical analysis on the relative intensity of LC3BII and P53 vs. GAPDH. GAPDH protein was used as loading control. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05 vs. OC group). (C) OC enhanced the binding between HSPA8, HSP90, and p53. A549 cells were treated with the combination of 10 µM OC, 40 µM cisplatin, or 10 µM PES for 24 h. Cell lysates were immunoprecipitated with HSPA8, and western blot analysis was performed for HSPA8, HSP90, and p53. (D) Statistical analysis on the relative intensity of HSP90 and P53 vs. HSPA8. HSPA8 protein was used as loading control. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. OC group). (E,F) OC potentiated cell death upon cisplatin treatment. A549 cells were treated with the combination of 10 µM OC, 40 µM cisplatin, or 10 µM PES for 24 h. The cells were collected and stained with Annexin V and PI for subsequent flow cytometry analysis. F shows the results of statistical analysis of apoptotic cells. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. OC group).

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Expressing, Western Blot, Control, Standard Deviation, Binding Assay, Immunoprecipitation, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: OC induces apoptosis of A549 cells by regulating HSPA8 protein. (A) Detection of the HSPA8 protein expression level in A549 cells by western blot analysis after 48 h of transfection, GAPDH was used as loading control. A549 cells were transfected with small interfering RNA (siRNA) and pCDNA_HSPA8 plasmids respectively for 48 h siRNA control (NC) and pCDNA3.1 vector (Vehicle) as a control. (B) The effects of OC on A549 cell viability after treatment with HSPA8 siRNA or pCDNA_HSPA8 plasmid. The cells were transfected with siRNA or plasmid for 48 h and OC was supplemented at the indicated concentrations for 72 h. Cell viability was then detected using the CCK-8 Assay. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). (C) OC induces apoptosis of A549 cells by targeting HSPA8 protein. A549 cells were transfected with HSPA8 siRNA or plasmid for 48 h, 10 µM OC was supplemented for another 24 h. The cells were collected and stained with Annexin V and PI for flow cytometry analysis. (D) The result of statistical analysis of apoptotic cells. Data are expressed as mean ± standard deviation of three independent experiments (** p < 0.01, *** p < 0.001 vs. OC control group).

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Expressing, Western Blot, Transfection, Control, Small Interfering RNA, Plasmid Preparation, CCK-8 Assay, Standard Deviation, Staining, Flow Cytometry