Journal: Journal of ovarian research

Article Title: Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

doi: 10.1186/s13048-023-01156-8

Figure Lengend Snippet: Fig. 1 The association of HSP60 with the prognosis of ovarian cancer patients and its expression in human ovarian cancer cell lines and tissues. A, B The curve plot for progression-free survival between HSPD1 high and low transcript groups in all OC and serous OC, respectively. The red line indicates patients with high HSPD1 levels and the black line indicates patients with low HSPD1 levels, log rank P = 0.01 and 0.00006, respectively. C Differential expression of HSP60 in the isolated mitochondria of OC cell lines. D Quantification analysis of the protein expression level in the C. E HSP60 expression was measured by IHC in tissue microarray. Representative images of strong (score 3), medium (score 2), weak (score 1), and negative (score 0) staining are shown. F IHC images of HSP60 in normal (upper) and OC (lower) tissues according to tissue microarray. G Statistical analysis of the protein expression of HSP60 according to the staining scores of OC and normal tissues. *P < 0.05, **P < 0.01

Article Snippet: Immunohistochemistry to detect HSP60 was performed using anti-HSP60 antibody (Proteintech, Wuhan, China; dilution 1:200).

Techniques: Expressing, Quantitative Proteomics, Isolation, Microarray, Staining

Journal: Journal of ovarian research

Article Title: Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

doi: 10.1186/s13048-023-01156-8

Figure Lengend Snippet: Fig. 2 Downregulation of HSPD1 promotes the proliferation ability of ovarian cancer cells. A Western blotting analysis of HSPD1 expression in SKOV3 and OVCAR3 cells transfected with NC siRNA and HSPD1 siRNA, respectively. B SKOV3 and OVCAR3 cells were transduced with lentiviruses delivering negative control shRNA (sh-NC) or HSPD1 shRNA (sh-HSPD1). Changes in HSPD1 protein expression was measured by western blotting. C Effects of HSPD1 knock down with siRNA on the proliferation of SKOV3 analyzed by the RTCA assay. D Effects of HSPD1 knockdown with lentivirus shRNA on the proliferation of SKOV3 analyzed by the RTCA assay. E Effects of HSPD1 knockdown with siRNA on the proliferation of OVCAR3 analyzed by the RTCA assay. F Graphical representation of the concentration of glucose in cell culture medium after 72 h cell culture. G Graphical representation of the concentration of lactate in cell culture medium after 72 h cell culture. *P < 0.05, ** P < 0.01 and ***P < 0.001

Article Snippet: Immunohistochemistry to detect HSP60 was performed using anti-HSP60 antibody (Proteintech, Wuhan, China; dilution 1:200).

Techniques: Western Blot, Expressing, Transfection, Transduction, Negative Control, shRNA, Knockdown, Concentration Assay, Cell Culture

Journal: Journal of ovarian research

Article Title: Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

doi: 10.1186/s13048-023-01156-8

Figure Lengend Snippet: Fig. 3 Knockdown of HSPD1 promoted ovarian cancer cell migration in vitro. A, B Effects of HSPD1 knockdown with siRNA on the migration of SKOV3 and OVCAR3 analyzed by transwell assay. The upper row are merged pictures. Scale bar = 50 μm. C Quantification of migrative cells in the si-NC and si-HSPD1 group. D, E Effects of HSPD1 knockdown with lentivirus shRNA on the migration of SKOV3 and OVCAR3 analyzed by transwell assay. Scale bar = 50 μm. F Quantification of migrative cells in the sh-NC and sh-HSPD1 group. *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Immunohistochemistry to detect HSP60 was performed using anti-HSP60 antibody (Proteintech, Wuhan, China; dilution 1:200).

Techniques: Knockdown, Migration, In Vitro, Transwell Assay, shRNA

Journal: Journal of ovarian research

Article Title: Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

doi: 10.1186/s13048-023-01156-8

Figure Lengend Snippet: Fig. 4 Identification of differentially expressed proteins in HSP60 knockdown SKOV3 cells via proteomics. A Heat map of Top 20 downregulated differen tially expressed proteins after HSP60 knockdown. B KEGG enrichment pathways of the Top 20 downregulated proteins

Article Snippet: Immunohistochemistry to detect HSP60 was performed using anti-HSP60 antibody (Proteintech, Wuhan, China; dilution 1:200).

Techniques: Knockdown

Journal: Journal of ovarian research

Article Title: Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

doi: 10.1186/s13048-023-01156-8

Figure Lengend Snippet: Fig. 5 HSP60 knockdown disrupted mitochondrial protein-OXSM stability and lipoic acid synthesis. A, B Western blotting analysis was used to validate expression changes of individual mitochondrial proteins in HSP60 knockdown SKOV3 cells with siRNA and lentivirus shRNA, respectively. C Quantitative RT-PCR analysis was used to analyze the mRNA levels of HSPD1, SUCLG2 and OXSM. D SKOV3 cells were transfected with si-NC and si-HSPD1, 48 h later, cells were treated with or without 0.2 µM MG132 for 24 h prior to cell lysis. The level of OXSM protein was examined by western blotting. E Western blot ting of lipoylated proteins (lip-DLAT) and total DLAT from extracts of OC cells with HSPD1 knockdown. F Kaplan-Meier survival analysis of progression- free survival between OXSM high and low transcript groups in all OC. The red line indicates patients with high OXSM levels and the black line indicates patients with low OXSM levels, log rank P = 0.01. ***P < 0.001

Article Snippet: Immunohistochemistry to detect HSP60 was performed using anti-HSP60 antibody (Proteintech, Wuhan, China; dilution 1:200).

Techniques: Knockdown, Western Blot, Expressing, shRNA, Quantitative RT-PCR, Transfection, Lysis

Journal: Journal of ovarian research

Article Title: Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM).

doi: 10.1186/s13048-023-01156-8

Figure Lengend Snippet: Fig. 6 HSPD1 promotes ovarian cancer progression by maintaining function of OXSM. A Co-immunoprecipitation of OXSM with HSP60 in SKOV3 cells. B Examination of interaction of OXSM-HSP60 PLA signal in SKOV3 and OVCAR3 cells lines. Scale bar = 25 μm. C Western blotting analysis of OXSM expression in SKOV3 cells transfected with NC siRNA and OXSM siRNA. D Validation of the overexpression of OXSM in OC cells transfected with OXSM plasmid via western blotting. E, G Effects of OXSM knockdown with siRNA on the proliferation of SKOV3 analyzed by the RTCA assay. F, H Effects of OXSM knockdown on the migration of SKOV3 analyzed by the RTCA assay. I, J Cell proliferation was detected by RTCA assay after transfection with sh-HSPD1 and OE-OXSM. Quantification of the cell index after 72 h culture in the RTCA assay. K, L SKOV3 cells transfected with lentivirus sh-NC or sh-HSPD1 treated with lipoic acid (0 or 0.125 µM). Quantification of the cell index after 72 h culture in the RTCA assay. *P < 0.05, **P < 0.01

Article Snippet: Immunohistochemistry to detect HSP60 was performed using anti-HSP60 antibody (Proteintech, Wuhan, China; dilution 1:200).

Techniques: Immunoprecipitation, Western Blot, Expressing, Transfection, Biomarker Discovery, Over Expression, Plasmid Preparation, Knockdown, Migration

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 2. Mass spectrometric analysis of the 14-3-3-binding protein in NTera2-N cells. Spot 1 labeled with the rh14-3-3z probe (Fig. 1) was excised from the gel, trypsinized, and processed for nanoESI-MS/MS analysis. (A) The spectra of MS analysis. Each peak indicates individual peptide fragments. The positions of several peaks were automatically numbered on the spectra. The fragments were selected for MS analysis in order of their signal intensity, although autolytic fragments of trypsin (e.g. 412, 421, 523, 737, 762, and 767) were omitted. (B) Amino acid sequence of human Hsp60. Seventeen peptide fragments of spot 1 identified by MS analysis (shadowed) showed a perfect match with the sequence encompassing amino acid residues 38–493 of human Hsp60. The number indicated on each fragment represents the position in the horizontal axis of the spectra.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Binding Assay, Labeling, Tandem Mass Spectroscopy, Sequencing

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 3. The interaction of 14-3-3 protein with Hsp60 was not zeta isoform-specific. Two hundred mi- crograms of total cellular protein of NTera2-N was separated on a 2- dimensional PAGE gel ([a, b]: pI 5.3– 6.3 and 4%–12%; [c, d]: pI 4–7 and 4%–12%), followed by silver stain- ing or phosphoprotein staining. The gel was processed for overlay with recombinant human 14-3-3g pro- tein (rh14-3-3g) tagged with Xpress, followed by relabeling with anti- Hsp60 antibody. (a) rh14-3-3z, (b) Hsp60, (c) silver staining, (d) phos- phoprotein staining. Spot 1 is in- dicated by an arrow.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Silver Staining, Staining, Recombinant

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 4. The interaction of 14-3-3 protein with Hsp60 was universal in neuronal and nonneuronal cells. Two hundred micrograms of total cellular protein of human cell lines and brain homogenate was separated on a 2-dimensional PAGE gel ([a–l]: pI 5.3–6.3 and 4% to 12%; [m–r]: pI 4-7 and 4% to 12%) followed by silver staining (the left panels). The gel was processed for overlay with the rh14-3-3z probe tagged with Xpress or Myc (the center panels) followed by relabeling with anti-Hsp60 antibody (the right panels). (a–c) Undifferentiated NTera2 teratocarcinoma (NTera2-U), (d–f) SK-N-SH neuroblastoma, (g–i) U-373MG astrocytoma, (j–l) HeLa cervical carcinoma, (m–o) human neuronal progenitor (NP) cells, (p–r) brain homogenate.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Silver Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 6. Colocalization of 14-3-3 protein, Hsp60, and PrPC in the mitochondria of human neuronal progenitor cells. Human neuronal progenitor (NP) cells were immunolabeled with 14-3-3z isoform-specific antibody, anti-Hsp60 antibody, anti-prion protein antibody (8G8), MitoTracker Red CMXRos, or DAPI followed by staining with secondary antibodies. (a) 14-3-3z, (b) Hsp60, (c) merge of (a) and (b); (d) 14-3-3z, (e) CMXRos, (f) merge of (d) and (e) labeled with DAPI; (g) PrPC, (h) Hsp60, (i) merge of (g) and (h).

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Immunolabeling, Staining, Labeling

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 8. Coexpression of 14-3-3 protein, Hsp60, and PrPC in reactive astrocytes in the human brain. Serial sections derived from autopsied brains of multiple sclerosis were processed for immunohistochemis- try using 14-3-3z isoform-specific antibody, anti-Hsp60 antibody, an- ti-prion protein antibody (8G8), or anti-GFAP antibody. (a–d) Chronic active demyelinating lesions in the subcortical white matter of the frontal lobe of 744 multiple sclerosis: (a) 14-3-3z, (b) Hsp60, (c) PrPC, (d) GFAP.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Derivative Assay

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 7. Coexpression of 14-3-3 protein, Hsp60, and PrPC in neurons in the human brain. Serial sections derived from autopsied brains of multiple sclerosis were processed for immunohistochemistry using 14-3-3z isoform-specific antibody, anti-Hsp60 antibody, anti-prion pro- tein antibody (8G8), or anti-neuron- specific enolase (NSE) antibody. (a– d) The cerebral cortex of the frontal lobe of #791 MS: (a) 14-3-3z, (b) Hsp60, (c) PrPC, (d) NSE.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Derivative Assay, Immunohistochemistry

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 9. A possible mechanism for elevation of 14-3-3 protein in the cerebrospinal fluid of prion diseases. When affected with the pathoge- netic prion, the molecular complex composed of 14-3-3, Hsp60, and PrPC becomes disintegrated during the conversion of PrPC into PrPSc aggregates, which displace the 14- 3-3 protein from the complex, resulting in the release of 14-3-3 from degenerating neurons into the cerebrospinal fluid.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability

doi: 10.1128/mcb.00203-19

Figure Lengend Snippet: Figure 8. ADAT3-V144M is bound by the HSP60 and TRiC chaperonins. (A) Silver stain of 1017

Article Snippet: Antibodies were against the following proteins : FLAG epitope tag 553 (A2220, Sigma), 6xHis tag (MA1-21315, Thermo Fisher), GFP (sc-9996, Santa Cruz 554 Biotechnology), Strep-tag II-tag (NC9261069, Thermo Fisher), ADAT3 (Abcam, ab192987), 555 ADAT3 (H00113179-B01P, Abnova), ADAT2 (ab135429, Abcam), HSP60 (A302-845A, 556 Bethyl Labs), CCT1 (sc-53454, Santa Cruz Biotechnologies), CCT7 (A304-730A-M, Bethyl 557 Labs) and actin (MAB1501, EMD Millipore).

Techniques: Silver Staining

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 1 Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. **p < 0.01 in comparison to control condition

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control, Comparison

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 2 HSP60 is indispensable for IL-1β-mediated NF-κB phosphorylation. a, b Effect of IL-1β was checked on phosphorylation of p65-NF-κB in the cytoplasmic extracts of N9 cells (a) and mice brain (b). c, d Role of HSP60 in the induction of phosphorylation of p65 was checked in N9 cells by overexpression of HSP60 (c) and knockdown of HSP60 (d). e Effect of HSP60 knockdown with vivo-morpholino was checked in mice brain after IL-1β treatment for 3 days. Representative blots of three independent experiments are shown here. Bar diagrams below the blots represent quantification of the relative fold changes in phosphorylation of p65-NF-κB in comparison to control. The levels of p-p65-NF-κB were normalized with total p65-NF-κB. *p < 0.05, **p < 0.01 in comparison to control values. ##p < 0.01 in comparison to IL-1β treatment. Data represented as means ±SD of three independent experiments

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Phospho-proteomics, Over Expression, Knockdown, Comparison, Control

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 3 HSP60 plays a critical role in IL-1β-induced nuclear localization of pNF-κB. a, d Effect of IL-1β was checked on the nuclear localization of phospho-p65-NF-κB in the N9 cells (a) and mice brain (d). b, c Role of HSP60 in the induction of phosphorylation of p65-NF-κB was checked in N9 cells by overexpression of HSP60 (b) and knockdown of HSP60 (c). e Effect of HSP60 knockdown using vivo-morpholino was checked on nuclear localization of p65-NF-κB in mice brain after IL-1β treatment for different time periods. The levels of p65-NF-κB were normalized with the nuclear loading control PCNA protein levels. Representative blots of three independent experiments are shown here. Bar diagrams below the blots represent quantification of the relative fold changes in phosphorylated levels of NF-κB in comparison to control. *p < 0.05, **p < 0.01 in comparison to control values. ##p < 0.01 in comparison to IL-1β treatment. Data represented as means ±SD of three independent experiments

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Phospho-proteomics, Over Expression, Knockdown, Control, Comparison

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 4 HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene (a–e) whereas the right panel shows the Western blot analysis (f–j). IL-1β treatment increased NLRP3 expression in vitro on both transcript level (a) and protein level (f). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR (d) and Western blotting (i). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level (b) and protein level (g). Effect of HSP60 knockdown on transcript levels (c, e) as well as protein levels (h, j) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. *p < 0.05, **p < 0.01 in comparison to control values. ##p < 0.01 in comparison to IL-1β treatment. Data represented as mean ± SD of three independent experiments

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, In Vivo, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 5 HSP60 induces mitochondrial damage and oxidative stress. a Mitochondrial damage was assessed in N9 cells using FACS by the quantification of mitochondrial membrane potential using Rhodamine 123 dye (upper panel). Histograms show the effect of IL-1β (i), effect of HSP60 overexpression (ii), and HSP60 knockdown (iii) on mitochondrial membrane potential. b ROS generation in N9 microglial cells was assessed by FACS using DCFDA (lower panel). Histograms in the lower panel show the effect of IL-1β (i), effect of HSP60 overexpression (ii), and HSP60 knockdown (iii) on ROS generation by microglia. Data show that HSP60 knockdown lead to significant reduction in mitochondrial depolarization and ROS generation by microglia (p < 0.01). For FACS analysis, each experiment was performed in triplicates. Results are representative of three independent experiments

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Membrane, Over Expression, Knockdown

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 6 Role of HSP60 in IL-1β-induced caspase-1 activation. Caspase-1 activity in the N9 cells (upper panel) and mice brain (lower panel) was assessed by caspase-1 activity kit. a Bar graphs in the upper panel show the effect of IL-1β (i), effect of HSP60 overexpression (ii), and HSP60 knockdown (iii) on caspase-1 activity in N9 cells. b Bar graphs in the lower panel show the effect of IL-1β (i) and HSP60 knockdown (ii) on caspase-1 activity in mice brain. Each experiment was performed in triplicates. Data represented as mean ± SD of three independent experiments (n = 3). *p < 0.05; **p < 0.01 in comparison to control values and ##p < 0.01 in comparison to IL-1β treatment

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Activation Assay, Activity Assay, Over Expression, Knockdown, Comparison, Control

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 7 HSP60 critically regulates microglial IL-1β production both in vitro and in vivo. Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene (a–e) while right panel shows the IL-1β ELISA (f–j). IL-1β treatment increases its own expression in vitro (a) and induces its own secretion also (f). Similarly, IL-1β expression was checked through qRT-PCR (d) and ELISA (i) in vivo. b, g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β (b) and its secretion from microglia (g). Effect of HSP60 knockdown on transcript levels (c, e) as well as secreted levels of IL-1β (h, j) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments (n = 3). *p < 0.05, **p < 0.01 in comparison to control values. ##p < 0.01 in comparison to IL-1β treatment. Data represented as mean ± SD of three independent experiments

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, Comparison, Control

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 8 Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level (a, b) and protein level (d, e) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f, g JEV infection increases IL-1β secretion both in vitro (f) and in vivo (g) which were analyzed using ELISA. h, i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells (h) and mice brain lysate (i). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments (n = 3). *p < 0.05, **p < 0.01 in comparison to control values and ##p < 0.01 with respect to JEV-infected values

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Virus, Quantitative RT-PCR, Infection, Western Blot, Expressing, Control, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Knockdown, Comparison

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 9 Downregulation of HSP60 reduces JEV-induced microglial inflammation. The left panel shows the effect of HSP60 knockdown with specific eSiRNA on JEV-induced microglial inflammation in N9 cells, while the right panel shows the effect of HSP60 knockdown using HSP60 vivo-morpholino in JEV-infected mice brains. a, b Western blots of iNOS and COX2 after HSP60 knockdown during JEV infection in N9 cells and mice brain respectively. Protein levels of iNOS and COX2 were normalized with the β-actin levels. The blots are representative of three independent experiments. c–h CBA of pro-inflammatory markers was performed to assess the role of HSP60 in JEV-induced microglial inflammation. Bar graphs show quantification of the cytokines levels in N9 cells (c–e) and in mice brains (f–h). Cytokine bead array was performed in triplicates for each experiment. For animal experiments, at least three mice were used in each group. Data represented as mean ± SD of three independent experiments (n = 3). *p < 0.05, **p < 0.01 in comparison to control values. ##p < 0.01 with respect to JEV-infected values

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Knockdown, esiRNA, Infection, Western Blot, Comparison, Control

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 10 Effect of HSP60 knockdown on the survival and behavior of the JEV-infected mice. a Survival plot showing increase in the survival of the mice after reduction in the inflammation by knockdown of HSP60. b Behavioral score plot shows delayed onset of the symptoms of JEV infection. Different scores were given for the behavior of the mice based on the symptoms. 0 = No pilorection; No body stiffening; No restriction of movement; No paralysis; No body tremor. 1 = Pilorection; No body stiffening; No restriction of movement; No paralysis; No body tremor. 2 = Pilorection; body stiffening; No restriction of movement; No paralysis; No body tremor. 3 = Pilorection; body stiffening; restriction of movement; No paralysis; No body tremor. 4 = Pilorection; body stiffening; restriction of movement; paralysis; No body tremor. 5 = Pilorection; body stiffening; restriction of movement; paralysis; body tremor. Data shown is representative of three different independent experiments and ‘n’ represents the number of animals in each group

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Knockdown, Infection

Journal: Journal of neuroinflammation

Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway.

doi: 10.1186/s12974-018-1214-5

Figure Lengend Snippet: Fig. 11 Schema of signaling pathway involved in HSP60-mediated NLRP3 inflammasome activation and subsequent IL-1β production. IL-1β induces its own production by the activated microglia in a HSP60-dependent manner. HSP60, after being upregulated by IL-1β, gets secreted outside and binds with TLR4 of the microglia to activate p38 MAPK [10]. Binding of HSP60 with TLR4 facilitates NF-κB phosphorylation, mitochondrial damage, and ROS generation and finally activates NLRP3 inflammasome leading to IL-1β production. JEV also augments HSP60 production and thus influences inflammasome complex to induce a consecutive expression of IL-1β and, in turn, induces an exaggerated immune response

Article Snippet: For the overexpression studies, 4 μg recombinant mouse HSP60 plasmid (MC206740, Origene) was used (Additional file 1: Figure S2), while 5 pM HSP60 eSiRNA (EMU151751, Sigma Aldrich) was used for knockdown experiments.

Techniques: Activation Assay, Binding Assay, Phospho-proteomics, Expressing