Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: Paracrine HSP27 promotes OSCC chemoresistance through TAMs. A Schematic presentation of the analyzed tumor models, Vehicle or HSP27-shRNA OSCC cells were orthotopically injected into BALB/c nude mice. B - C Tumor pictures ( B ) and growth analysis ( C ) over 21 days show the mean tumor volume at the indicated time points following implantation, statistical significance was determined by 2-way ANOVA. Volume of individual tumors at day 21 is shown in the box plot; statistical significance was determined by Welch t-test. D - E HSP27 mRNA ( D ), protein ( E ) levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR and Western blotting in the whole-cell lysates. F Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR. G Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by RT-qPCR. H Relative cell apoptosis level in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Flow Cytometry. I Relative cell apoptosis level in CAL27 cells without or with rhHSP27 (2μg/mL) induced TAMs-CM, detected by Flow Cytometry. J Analysis of ( H ), determined with relative ratio of apoptosis in total per 10 4 cells. K Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Western blotting in the whole-cell lysates. L Analysis of ( I ), determined with relative ratio of apoptosis in total per 10 . 4 cells. M Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by Western blotting in the whole-cell lysates. N – O Analysis of ( K ) and ( M ). T-tests and two-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: shRNA, Injection, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: Paracrine HSP27 promotes OSCC invasion, migration, and EMT through TAMs. A GSEA of transcriptome data cells shows enrichment of a gene signature associated with increased metastasis in HNSCC. B The correlation between the expression of HSP27 and the metastasis pathway enrichment score. C The EMT pathway enrichment score of HSP27 and TLR4. D Representative invasive (upper) and migratory (lower) images of the transwell assays using CAL27 cells (upper chamber) cocultured with TAMs (lower chamber) and preincubated in the presence of rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM). E Representative images of the wound-healing assays using CAL27 cells under the stimulation of TAMs-CM from TAMs preincubated with rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM). F – H statistical analysis of cell invasion ( F ), migration ( G ), and stretch ( H ). I Representative immunofluorescence staining images showing the colocation of HSP27 and TLR4 on the cell membranes of the RAW 264.7 cells incubated with rhHSP27 (2 µg/mL) in the TCM of the CAL27 cells. Nuclei were counterstained with DAPI. J - L N-cadherin, Vimentin mRNA and N-cadherin protein levels in the CAL27 cells under stimulation with the TAMs-CM from the TAMs incubated in the presence of rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM), detected by RT-qPCR and Western blotting. One-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: Migration, Expressing, Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: HSP27/IL-6 establishes a positive feedback loop between OSCC cells and M2 TAMs. A - B iNOS, IL-12, TNF-α, Arg-1, IL-10, and IL-6 mRNA levels in RAW264.7cells under stimulation with the TCM of the CAL27 cells pretreated without or with HSP27–siRNA, detected by RT-qPCR. C - D iNOS, IL-12, Arg-1, CD163, and IL-6 mRNA levels in RAW264.7cells pretreated without or with rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR. E – F IL-12, Arg-1, and IL-6 mRNA and IL-6 protein levels in RAW264.7cells pretreated without or with TAK-242 (1 µM) under the stimulation of rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR ( E ) and ELISA ( F ). G - H IL-6 mRNA, protein levels in THP-1 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR ( G ) and ELISA ( H ). I - J IL-6 mRNA, protein levels in THP-1 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR ( I ) and ELISA ( J ). K - L IL-6 mRNA, protein levels in RAW264.7 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR ( K ) and ELISA ( L ). M – N ), IL-6 mRNA, protein levels in Raw264.7 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR ( M ) and ELISA ( N ). O - P HSP27 mRNA levels in CAL27 cells without or with IL-6 (100 ng/mL) treated ( O ), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated ( P ), detected by RT-qPCR. ( Q - S ), HSP27protein levels in CAL27 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates ( Q ) and ELISA ( R - S ). T - U HSP27 mRNA levels in SCC9 cells without or with IL-6 (100 ng/mL) treated ( T ), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated ( U ), detected by RT-qPCR. V-X HSP27 protein levels in SCC9 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates ( V ) and ELISA ( W - X ). T-tests and one-way ANOVA. Means ± SD of at least three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Primary mouse astrocytes were treated for 24 hours either with conditioned medium from astrocytes transfected with V5-hHSPB1 or with rhHSPB1. Created with BioRender.com . ( B ) Anti-V5 and anti-human HSPB1 (hHSPB1) antibodies were used to detect the levels of V5-hHSPB1 in astrocyte lysates exposed to conditioned medium for 24 hours. ( C ) An anti-hHSPB1 antibody was used to detect the levels of human HSPB1 in astrocytes treated with the indicated concentrations of rhHSPB1 for 24 hours or with heat-inactivated (HI) rhHSPB1. ( D ) Confocal live imaging of astrocytes exposed to rhHSPB1 labeled with Oregon Green for 24 hours and stained with lysotracker. ( E ) Primary mouse neurons were treated with either conditioned medium from astrocytes transfected with V5-hHSPB1 and grown in Neurobasal medium or with rhHSPB1. Created with BioRender.com . ( F ) Anti-V5 and anti-hHSPB1 antibodies were used to detect the levels of V5-hHSPB1 in neuron lysates exposed to conditioned medium for 8 hours. ( G ) An anti-hHSPB1 antibody was used to detect the levels of human HSPB1 in neurons treated with the indicated concentrations of rhHSPB1, or heat-inactivated rhHSPB1, for 24 hours. Equivalent amounts of rhHSPB1 in control or upon heat inactivation were run in parallel to confirm its detection by Western blotting. Note that the background detected in neuron lysates at 0 ng/ml is unspecific since (i) neurons in culture do not express HSPB1, as we have shown in , and (ii) human HSPB1 antibody does not recognize the mouse HSPB1 [as shown in (C), where astrocytes express high levels of murine HSPB1]. ( H ) Confocal live imaging of neurons exposed for 6 hours to rhHSPB1 labeled with Oregon Green and stained with lysotracker.

Article Snippet: Neurons at 6 DIV were transduced with AAVs to express wild-type (AAV2/TM8-WT-htau0N4R-EGFP) or mutant (AAV2/TM8-P301L/S320F-htau0N4R-EGFP) htau at 2.5 × 10 10 viral genome (VG)s/ml, followed by treatment with either rhHSPB1 (50 ng/ml in 0.1% BSA, R&D Systems, 1580-HS) or vehicle (0.1% BSA in PBS), at 6 DIV and every 48 hours until 12 DIV.

Techniques: Transfection, Imaging, Labeling, Staining, Control, Western Blot

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Primary astrocytes were pretreated with rhHSPB1 for 24 hours before treatment with either 0.1% BSA (vehicle) or rhHSPB1 (50 ng/ml) and either TNFα + IL-1α or control for further 24 hours. Created with BioRender.com . ( B ) LCN2, SerpinA3N, and iNOS were detected by Western blotting in astrocyte lysates, and levels were quantified relative to GADPH as a loading control ( C to E ). ( F and G ) LCN2 was detected in concentrated conditioned medium and quantified relative to intracellular GAPDH levels (G). ( H ) The concentration of nitrites in the astrocyte medium was measured as an indirect measure of NO using a Griess assay. ( I ) The levels of IL-6 in the astrocyte medium were determined by ELISA. ( J and K ) A cytokine array was used to determine levels of 38 cytokines in the medium in a membrane-based sandwich immunoassay. The average z scores for each of the 38 cytokines was represented in the graph (J) or shown individually for each cytokine in the heatmap (K), where cytokines are ranked from low to high fold change of TNFα + IL-1α/rhHSPB1 versus TNFα + IL-1α/vehicle. Cytokines with an asterisk showed significant increase (red) or decrease (green) in TNFα + IL-1α/rhHSPB1 versus TNFα + IL-1α/vehicle condition. Data are shown as means ± SD ( N = 3). Two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test [(C), (D), (E), (G), (H), and (J)] or unpaired Student’s t test (I). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Neurons at 6 DIV were transduced with AAVs to express wild-type (AAV2/TM8-WT-htau0N4R-EGFP) or mutant (AAV2/TM8-P301L/S320F-htau0N4R-EGFP) htau at 2.5 × 10 10 viral genome (VG)s/ml, followed by treatment with either rhHSPB1 (50 ng/ml in 0.1% BSA, R&D Systems, 1580-HS) or vehicle (0.1% BSA in PBS), at 6 DIV and every 48 hours until 12 DIV.

Techniques: Control, Western Blot, Concentration Assay, Griess Assay, Enzyme-linked Immunosorbent Assay, Membrane

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Organotypic brain slices were prepared from postnatal mouse brain, transduced at 0 DIV with AAVs to express hHSPB1 in astrocytes under the GFAP promoter for 20 DIV followed by treatment with TNFα + IL-1α for 24 hours. Created with BioRender.com . ( B ) Transduction of slices with AAV-GFAP-BFP (AAV-GFAP) and AAV-GFAP-hHSPB1:BFP (AAV-GFAP-hHSPB1) shows astrocyte-specific (GFAP + ) expression of BFP and hHSPB1 (note that HSPB1 antibody has some unspecific nuclear staining). ( C ) Western blotting to confirm expression of hHSPB1 and its secretion in brain slices. ( D to F ) Changes in LCN2 were quantified in lysates (E) and in conditioned medium (F) relative to intracellular GAPDH. ( G and H ). A cytokine array measured levels of 38 cytokines in the medium in a membrane-based sandwich immunoassay. The average z scores for each cytokine were represented in the graph (G) or shown individually in the heatmap (H), where cytokines are ranked from high to low fold change of control/AAV-GFAP versus TNFα + IL-1α/AAV-GFAP. Cytokines marked with a green asterisk were significantly decreased in TNFα + IL-1α/AAV-GFAP-hHSPB1 versus TNFα + IL-1α/AAV-GFAP conditions. ( I ) The hippocampus was separated and cultured for 19 days and then pretreated with rhHSPB1 (50 ng/ml) for 24 hours and with either control or TNFα + IL-1α and either vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml) for further 24 hours. ( J ) Levels of LCN2 were detected by immunoblot in slice lysates and quantified relative to GAPDH ( K ). Data are shown as means ± SD. N = 5 [(E) and (F)], N = 3 [(G) and (H)], or N = 6 [(J) and (K)]. Two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001.

Article Snippet: Neurons at 6 DIV were transduced with AAVs to express wild-type (AAV2/TM8-WT-htau0N4R-EGFP) or mutant (AAV2/TM8-P301L/S320F-htau0N4R-EGFP) htau at 2.5 × 10 10 viral genome (VG)s/ml, followed by treatment with either rhHSPB1 (50 ng/ml in 0.1% BSA, R&D Systems, 1580-HS) or vehicle (0.1% BSA in PBS), at 6 DIV and every 48 hours until 12 DIV.

Techniques: Transduction, Expressing, Staining, Western Blot, Membrane, Control, Cell Culture

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Primary mouse neurons were treated with rhHSPB1 (10 ng/ml) or vehicle (0.1% BSA) every 3 days from 3 DIV, fixed on 14 DIV, and stained for MAP2. ( B to G ) High-content confocal microscopy was used to determine changes in the number of segments (B), end points (C), primary branching points (D), secondary branching points (E), root points (F) per neuron, and the total length of all neurites for each neuron (G) ( N = 6). ( H ) Neurons were treated with conditioned medium from control or TNFα + IL-1α–treated astrocytes at 10 and 12 DIV, together with rhHSPB1 (50 ng/ml) or vehicle (0.1% BSA), and the medium was collected at 13 DIV. In parallel, neurons were treated with equivalent concentrations of TNFα + IL-1α. Cellular toxicity was determined by measuring LDH levels in neuron-conditioned medium ( N = 5). ( I ) Neurons were transduced at 6 DIV with AAVs to express WT-htau-EGFP or mutant P301L/S320F-htau-EGFP and treated with either vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml) every 2 days and fixed at 12 DIV. Created with BioRender.com . ( J ) Representative images of neurons transduced with mutant P301L/S320F-htau-EGFP and treated with vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml). ( K ) Graph shows the percentage of EGFP-positive neurons that show mutant htau aggregates. N = 6 with >1500 cells counted per experiment. ( L and M ) Total levels of tau were detected using an antibody that recognizes total tau, and the band corresponding to htau tagged to EGFP was quantified relative to β-actin. ( N ) Cellular toxicity was determined by measuring LDH levels in neuron-conditioned medium ( N = 4). Data are shown as means ± SD. Paired Student’s t test [(B) to (G) and (K)]; two-way ANOVA with Tukey’s multiple comparisons test [(H), (M), and (N)], * P < 0.05, ** P < 0.01.

Article Snippet: Neurons at 6 DIV were transduced with AAVs to express wild-type (AAV2/TM8-WT-htau0N4R-EGFP) or mutant (AAV2/TM8-P301L/S320F-htau0N4R-EGFP) htau at 2.5 × 10 10 viral genome (VG)s/ml, followed by treatment with either rhHSPB1 (50 ng/ml in 0.1% BSA, R&D Systems, 1580-HS) or vehicle (0.1% BSA in PBS), at 6 DIV and every 48 hours until 12 DIV.

Techniques: Staining, Confocal Microscopy, Control, Mutagenesis, Transduction

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Organotypic brain slices were transduced with AAVs to express WT-htau-EGFP or mutant P301L/S320F-htau-EGFP and (i) simultaneously transduced with AAV-GFAP or AAV-GFAP-hHSPB1 or (ii) treated with either vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml) from 14 DIV and then every 2 to 3 days up to 28 DIV. Created with BioRender.com . ( B ) Representative images of slices with AAV-WT-htau-EGFP or AAV-P301L/S320F-htau-EGFP. ( C to E ) Total lysates from AAV-GFAP-HSPB1 or control transduced slices were subjected to sarkosyl extraction to isolate the low-speed supernatant (total tau), high-speed supernatant (soluble tau), or sarkosyl-insoluble pellet (insoluble tau). Tau (D) or Ser 396 /Ser 404 phosphorylated tau (PHF-1) (E) were quantified in the whole lane (between 15 and 250 kDa) to include both mouse (mtau) and htau and any modified tau, and quantified relative to β-actin or total tau, as indicated. ( F to H ) Lysates from brain slices treated with rhHSPB1 or vehicle were subjected to sarkosyl extraction, and levels of tau (G) or PHF-1 (H) were quantified as above. ( I ) Intracellular LCN2 was detected in lysates from slices transduced with AAV-WT-htau-EGFP or AAV-P301L/S320F-htau-EGFP and AAV-GFAP-HSPB1 or control at 0 DIV and collected at 28 DIV. Levels of LCN2 relative to ALDH1L1 are shown ( J ). Data are shown as means± SD. N = 6 [(D) and (E)], N = 5 [(G) and (H)], or N = 3 (J). Two-way ANOVA with Tukey’s multiple comparisons test [(D), (E), (G), (H), and (J)], * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( K ) Working model showing that reactive astrocytes that surround amyloid plaques or that are exposed to proinflammatory cytokines from activated microglia secrete HSPB1 into the extracellular space. Secreted HSPB1 can have autocrine functions by ameliorating the reactive inflammatory response in astrocytes, as well as paracrine functions by reducing tau inclusion pathology and promoting neuronal health in adjacent neurons. Created with BioRender.com .

Article Snippet: Neurons at 6 DIV were transduced with AAVs to express wild-type (AAV2/TM8-WT-htau0N4R-EGFP) or mutant (AAV2/TM8-P301L/S320F-htau0N4R-EGFP) htau at 2.5 × 10 10 viral genome (VG)s/ml, followed by treatment with either rhHSPB1 (50 ng/ml in 0.1% BSA, R&D Systems, 1580-HS) or vehicle (0.1% BSA in PBS), at 6 DIV and every 48 hours until 12 DIV.

Techniques: Transduction, Mutagenesis, Control, Extraction, Modification

Journal: bioRxiv

Article Title: Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

doi: 10.1101/2021.10.29.466491

Figure Lengend Snippet: HBMEC cells were cultured in the 96-well plates until 80% confluency in a humidified incubator. ( a-c ) Confluent monolayers were treated with EXOs, MVs, and EVs (EXO: MV 1:1) at the indicated amounts for 24 ( a ), 48 ( b ), and 72h ( c ). Polyethyleneimine, PEI, at 50 µg/mL was used as a positive control for the ATP assay. Post-treatment, cells were incubated with a 1:1 mixture of fresh growth medium and Cell titer Glo reagent. The relative luminescence units (RLU) of the samples were measured using a SYNERGY HTX multimode plate reader at 1s integration time. Relative ATP levels were calculated by normalizing the RLU of treatment groups to the RLU of control, untreated cells. ( d ) Confluent normoxic HBMEC monolayers were treated with OGD medium and incubated in a hypoxic Billups-Rothenberg chamber at 37°C for 24 h. The following groups were also included: Normoxic HBMECs cultured in complete growth medium in a humidified incubator, oxygen-deprived cells cultured in complete growth medium in a hypoxic chamber, glucose-deprived cells cultured in OGD medium in a normoxic chamber. The cell viability of normoxic HBMECs was used as a control to calculate the cell viability in all treatment conditions. ( e ) Confluent HBMECs were treated with the indicated doses of EXOs and MVs in OGD medium and cell viability was measured 24 h post-treatment while untreated cells were used as a control. ( f ) Normoxic confluent HBMECs were treated with the indicated samples at a dose of 2 µg of HSP27 per well for 72 h before measuring cell viability. Data represent mean ±SD (n=3). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Article Snippet: Recombinant human HSP27 was purchased from Novus Biologicals (Centennial, CO).

Techniques: Cell Culture, Positive Control, ATP Assay, Incubation, Control

Journal: bioRxiv

Article Title: Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

doi: 10.1101/2021.10.29.466491

Figure Lengend Snippet: (a) Native polyacrylamide gel electrophoresis (PAGE) for PEG-DET/HSP27 complexes. Native HSP27, PEG-DET/HSP27 at indicated weight ratios, and free PEG-DET polymers were mixed with 1x native sample buffer and loaded in an SDS-free 4-10% polyacrylamide gel at 1 µg HSP27 per lane. (c,e) Native PAGE for hCMEC/D3-derived EV/HSP27 complexes: Native HSP27 and mixtures of EXO/HSP27 ( c ), and MV/HSP27 ( e ) at 5, 10, and 15:1 weight/weight (w/w) ratios were loaded in a SDS-free 4-10% polyacrylamide gel at 1 µg HSP27 per lane. Free EXOs and MVs equivalent to the amounts in 5:1, 10:1, and 15:1 w/w complexes were used as controls. ( g ) native PAGE for (PEG-DET/HSP27)/EV ternary mixtures. PEG-DET/HSP27 complexes were prepared at 20:1 and 30:1 w/w ratios followed by incubation with 10 µg of EVs. The indicated samples were loaded in the gel at 1 µg HSP27/lane. Each gel was run at 100 V for 2 h and stained using Biosafe Coomassie G250. The gel was then scanned at 800 nm using an Odyssey imager at intensity setting 5. ( b,d,f,h ) Densitometry analysis was performed by measuring band densities of HSP27 in the different experimental groups in comparison to the band density of native HSP27 in the respective gel using Image Studio 5.0 software *p<0.05

Article Snippet: Recombinant human HSP27 was purchased from Novus Biologicals (Centennial, CO).

Techniques: Polyacrylamide Gel Electrophoresis, Clear Native PAGE, Derivative Assay, Incubation, Staining, Comparison, Software

Journal: bioRxiv

Article Title: Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

doi: 10.1101/2021.10.29.466491

Figure Lengend Snippet: Average particle diameters ( a ), dispersity indices ( b ), and zeta potentials ( c ) of the indicated samples were measured using dynamic light scattering on a Malvern Zetasizer Pro-Red. Average particle diameters ( d ), dispersity indices ( e ), and zeta potentials ( f ) of PEG-DET/EV and (PEG-DET/HSP27)/EV complexes at the indicated weight ratios. The samples containing 1 µg HSP27 protein were diluted to 50 µL in 10 mM HEPES buffer pH 7.4 for particle diameter measurements. The diluted samples were further diluted to 800 µL in 10 mM HEPES buffer pH 7.4 for zeta potential measurements. Data represent mean±SD (n=3). * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: Recombinant human HSP27 was purchased from Novus Biologicals (Centennial, CO).

Techniques: Zeta Potential Analyzer

Journal: PloS one

Article Title: Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

doi: 10.1371/journal.pone.0066001

Figure Lengend Snippet: Figure 1. Characterization of hHSP27. A–B, Isolated human heat shock protein 27 (hHSP27) or recombinant HSP27 (rHSP27) proteins (1 mg or 10 mg) were separated by SDS-PAGE (A) and native-PAGE (B) and stained with Coomassie brilliant blue. C, hHSP27 proteins were immunoblotted with antibodies against HSP27, phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27. D, hHSP27 proteins were separated by native-PAGE and immunoblotted with antibodies against ab-crystallin and HSP20. E, rHSP27 (10 ng), hHSP27 (10 ng), ab-crystallin (5 ng), and HSP20 (5 ng) were separated by SDS-PAGE followed by immunoblotting with antibodies against HSP27, ab-crystallin, and HSP20. doi:10.1371/journal.pone.0066001.g001

Article Snippet: The observers of the behavioral tests were blinded to the treatment groups, and mice of the various groups were randomized during a given testing period. hHSP27 and HSP27 Antibody, HSP27 Elution Peptide Administration or Recombinant HSP27 Administration Mice received intravenous injections of 50 mg of hHSP27 mixed with 50 or 500 mg of HSP27-N1 or -C1 antibody 1 h after reperfusion (n= 3 in each group), 5 or 50 mg of HSP27-N1 and - C1 peptides, which were used in the elution, intravenously 1 h after reperfusion (n= 3 in each group), or 50 mg of recombinant HSP27 (rHSP27; Acris Antibodies GmbH) 1 h after reperfusion (n= 3).

Techniques: Isolation, Recombinant, SDS Page, Clear Native PAGE, Staining, Western Blot

Journal: PloS one

Article Title: Human-derived physiological heat shock protein 27 complex protects brain after focal cerebral ischemia in mice.

doi: 10.1371/journal.pone.0066001

Figure Lengend Snippet: Figure 4. Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects. A, Infarct volumes in control, hHSP27 (50 mg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means6SEM of 3 mice in each group. **P,0.001 vs. controls. B– D, Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDS- PAGE (B) and native-PAGE (C), stained with Coomassie brilliant blue (B,C), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (D). E, Photomicrographs of infarct areas stained with cresyl violet in hHSP27 and dephosphorylated hHSP27 groups prepared 24 h after reperfusion. Scale bar = 1 mm. hHSP27, human heat shock protein. doi:10.1371/journal.pone.0066001.g004

Article Snippet: The observers of the behavioral tests were blinded to the treatment groups, and mice of the various groups were randomized during a given testing period. hHSP27 and HSP27 Antibody, HSP27 Elution Peptide Administration or Recombinant HSP27 Administration Mice received intravenous injections of 50 mg of hHSP27 mixed with 50 or 500 mg of HSP27-N1 or -C1 antibody 1 h after reperfusion (n= 3 in each group), 5 or 50 mg of HSP27-N1 and - C1 peptides, which were used in the elution, intravenously 1 h after reperfusion (n= 3 in each group), or 50 mg of recombinant HSP27 (rHSP27; Acris Antibodies GmbH) 1 h after reperfusion (n= 3).

Techniques: De-Phosphorylation Assay, Control, Recombinant, SDS Page, Clear Native PAGE, Staining