Journal: ACS Chemical Neuroscience

Article Title: Astrocyte Mitochondria Are a Sensitive Target of PCB52 and its Human-Relevant Metabolites

doi: 10.1021/acschemneuro.4c00116

Figure Lengend Snippet: HSP60-stained C6 cells following exposure to PCB52 and its metabolites. (A) C6 cells were exposed to PCB52 and its human-relevant metabolites for 2 h and then fixed, immunostained for HSP60, a mitochondrial matrix protein, and imaged to visualize the mitochondrial structure. Data analysis of confocal images showed that PCB52 and both the metabolites (B) increased mitochondrial number and significantly decreased (C) mitochondrial length, (D) form factor, and (E) aspect ratio. (F) XY plot of the two independent metrics, the form factor and aspect ratio, indicating both metrics change in the same direction. n = 3 with 2 technical replicates per “ n ”, at least 10 images per “ n ” were analyzed (at least ∼30 astrocytes per exposure condition); scale bar, 10 μm; data were analyzed using one-way ANOVA with post hoc Dunnett’s test, p values <0.05 indicating statistical significance are shown in the figure.

Article Snippet: The Proteintech anti-HSP60 antibody (Cat. no. 66041-1-Ig), provided by Dr. Strack, was diluted at a ratio of 1:500 in blocking solution.

Techniques: Staining

Journal: bioRxiv

Article Title: Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting

doi: 10.1101/2021.03.09.434573

Figure Lengend Snippet: A) Fitness score for HSPD1 in lung adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). Each dot represents a single gene. B) RSA (redundant siRNA activity) waterfall plot of HSPD1 sensitivity score from PROJECTDRIVE showing HSPD1 as an essential gene in non-small cell lung cancer (NSCLC) cell lines (n=52). RSA sensitivity score <-3. C) Overall survival analysis between the low- and high-HSPD1 groups in TCGA LUAD (N=510) and GSE30219 (N=293). P-value was calculated using log-rank test. Relapse-free (D) and disease-specific (E) survival analysis between low- and high-HSPD1 groups in GSE30219 (N=293) and TCGA LUAD (N=510), respectively. P-value was calculated using log-rank test. Time is expressed as months. IHC staining of HSPD1 in cancer tissues (F, G, H and I) derived from consecutive 20 lung cancer patients (peritumoral bronchial structure in upper right corner of panel F). J) Pie chart of HSPD1 predominant intensity in patient-derived adenocarcinoma samples. K) Western blot quantification of HSPD1 in a panel of different human (A549, Calu-1, SK-MES-1, H460, H520, H1299 and H23) and mouse (Ladi2.1, LL2 and Ladi3.1) NSCLC cells. β-Actin was used as loading control. L) Western blot analysis of HSPD1 in cytosolic (named as C) or mitochondrial (named as M) fraction. β-Actin was used as loading control and TOMM20 was used as control for cytosolic/mitochondrial fractionation.

Article Snippet: Briefly, samples were pretreated for 36 minutes with antigen retrieval ULTRA CC1 then they were incubated for 32 minutes at 36°C with HSP60 (HSPD1) (AbTA800758, Clone OTI3A2, 1:100 dilution, ORIGENE, Rockville, US) primary antibody.

Techniques: Activity Assay, Immunohistochemistry, Derivative Assay, Western Blot, Fractionation

Journal: bioRxiv

Article Title: Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting

doi: 10.1101/2021.03.09.434573

Figure Lengend Snippet: A) Western blot analysis of HSPD1 protein levels in A549, H1299 and H460 cells upon infection with 3 independent shRNA (#44, #45 and #48) targeting HSPD1 compared to scramble-infected cells (pLKO). β-Actin was used as loading control. B) Real-time proliferation curves of A549, H1299 and H460 infected with non-targeting pLKO or shHSPD1. Plotted is the cells’ confluency over time. P-values are from two-way ANOVA. Points (n=5) are average ± SD. *<0.05, **<0.01, ***<0.001, ****<0.0001. Colony formation of A549 (C) and H1299 (D) cells with pLKO or shHSPD1, stained with crystal-violet and quantified in triplicates. Scale bars are average ± SD. P-values are from unpaired t -test. **<0.01, ***<0.001. FACS plots of A549 (E) and H1299 (F) cells infected with pLKO or shHSPD1 and stained with PI for cell cycle analysis. Bar graph showing the % of cells in each cell cycle phase of A549 (G) and H1299 (H) cells upon infection with pLKO or shHSPD1. P-values are from two-way ANOVA. Scale bars (n=3) are average ± SD. **<0.01, ***<0.001.

Article Snippet: Briefly, samples were pretreated for 36 minutes with antigen retrieval ULTRA CC1 then they were incubated for 32 minutes at 36°C with HSP60 (HSPD1) (AbTA800758, Clone OTI3A2, 1:100 dilution, ORIGENE, Rockville, US) primary antibody.

Techniques: Western Blot, Infection, shRNA, Staining, Cell Cycle Assay

Journal: bioRxiv

Article Title: Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting

doi: 10.1101/2021.03.09.434573

Figure Lengend Snippet: A) Western blot analysis of HSPD1 protein level in SK-MES-1 and Calu-1 cells upon infection with respectively 5 or 3 independent shRNAs (#44, #45 #46, #47 and #48) targeting HSPD1 compared to scramble-infected cells (pLKO). β-Actin was used as loading control. B) Real-time proliferation curves of SK-MES-1 and Calu-1 infected with non-targeting pLKO or shHSPD1. Plotted is cells’ confluency over time. P-values are from two-way ANOVA. Points (n=5) are average ± SD. ****<0.0001. Colony formation of H460 (C) and Calu-1 (D) cells infected with pLKO or shHSPD1, stained with crystal-violet and quantified in triplicates. Scale bars are average ± SD. P-values are from unpaired t -test. **<0.01, ***<0.001. E) FACS plots of Calu-1 cells infected with pLKO or shHSPD1 and stained with PI for cell cycle analysis. F) Bar graph showing the % of cells in each cell cycle phase of Calu-1 cells upon infection with pLKO or shHSPD1. P-values are from two-way ANOVA. Scale bars (n=3) are average ± SD. *<0.05, ****<0.0001.

Article Snippet: Briefly, samples were pretreated for 36 minutes with antigen retrieval ULTRA CC1 then they were incubated for 32 minutes at 36°C with HSP60 (HSPD1) (AbTA800758, Clone OTI3A2, 1:100 dilution, ORIGENE, Rockville, US) primary antibody.

Techniques: Western Blot, Infection, Staining, Cell Cycle Assay

Journal: bioRxiv

Article Title: Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting

doi: 10.1101/2021.03.09.434573

Figure Lengend Snippet: A) Bar graph showing IC 50 values (μM) of all the 26 NSCLC cells belonging to CL-100 ProLiFiler screenin and dose-response curves (normalized to DMSO control) to KHS101 of 2 cell lines. Points (n=2) are average ± SD. IC 50 values (μM) are shown with 95% confidence intervals. B) Bar graph showing the average IC 50 for each NSCLC histotype and SCLC. Bars are average ± SD. P-values are from one-way ANOVA. C) Dose-response curves of the 4 more sensitive cell lines (H460, NCI-H1581, LOU-NH91, A549) compared to the 4 more resistant cell lines (NCI-H838, BEN, NCI-H1563, SK-LU-1) resulted from the screening. Points are average ± SD. D) Correlation between HSPD1 gene expression and KHS101 IC 50 values in a panel of NSCLC cell lines from GSE47206 (n=13). E) Genes with most differential expression between KHS101-sensitive and KHS101-resistant groups (log 2 fold change cut-off of 4 and p-value<0.05) F) Western blot analysis of NLRC5 protein level in A549 cells overexpressing NLRC5 or empty vector. β-Actin was used as loading control. G) Dose-response curves (normalized to DMSO control) of A549 expressing NLRC5 compared to empty control cells. Points (n=4) are average ± SD. IC 50 values (μM) are shown. P-values are from two-way ANOVA. ****<0.0001. H) Dose-response curves (normalized to DMSO control) of A549 SLC6A8 KO compared to control cells. Points (n=4) are average ± SD. IC 50 values (μM) are shown. P-values are from two-way ANOVA. ****<0.0001 I) Metabolite analysis between KHS101-sensitive and KHS101-resistant cells with a log 10 fold change of 2 and p-value<0.05. J) Metabolic phenogram showing OCR (basal respiration) and ECAR (indicative glycolysis) values of creatine high (H460, A549) and creatine low (BEN, H838) cells. K) Metabolic phenogram showing OCR and ECAR levels in A549 treated for 24 hours with creatine-kinase inhibitor (DNFB). L) Dose-response curves (normalized to DMSO control) of A549 and H460 cells in presence of creatine-kinase inhibitor DNFB. Points (n=3) are average ± SD. IC 50 values (μM) are shown. P-values are from two-way ANOVA. *<0.05, ***<0.001.

Article Snippet: Briefly, samples were pretreated for 36 minutes with antigen retrieval ULTRA CC1 then they were incubated for 32 minutes at 36°C with HSP60 (HSPD1) (AbTA800758, Clone OTI3A2, 1:100 dilution, ORIGENE, Rockville, US) primary antibody.

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting

doi: 10.1101/2021.03.09.434573

Figure Lengend Snippet: A) Scheme showing the genome-wide CRISPR-Cas9 screening platform employed to identify KHS101 resistance genes. B) Gene ontology (GO) analysis of the top 20 candidates identified from the knockout screen in cells after treatment with KHS101. GO was performed with the cellular component 2017b gene set library in Enrichr tool. C) Dose-response curves to KHS101 (normalized to DMSO control) of A549 cells overexpressing two gRNAs to knockout COX5B, compared to parental A549. Points (n=4) are average ± SD. IC 50 values (μM) are shown. P-values are from two-way ANOVA. *<0.05. D) Bar graphs showing quantification of basal respiration, indicative glycolysis and ATP-linked respiration of A549 COX5B knockout cells compared to parental cells. Bars (n=5) are average ± SD. P-values are from unpaired t -test. ***<0.001, ****<0.0001. E) Dose-response curves (normalized to DMSO control) of A549, H460 and H1299 cells in presence of glycolysis inhibitor 2-deoxy-D-glucose (2-DG). Points (n=3) are average ± SD. IC 50 values (μM) are shown. P-values are from two-way ANOVA. ****<0.0001. F) Schematic representation of the metabolic alteration that occur in NSCLC cells upon HSPD1 loss.

Article Snippet: Briefly, samples were pretreated for 36 minutes with antigen retrieval ULTRA CC1 then they were incubated for 32 minutes at 36°C with HSP60 (HSPD1) (AbTA800758, Clone OTI3A2, 1:100 dilution, ORIGENE, Rockville, US) primary antibody.

Techniques: Genome Wide, CRISPR, Knock-Out

Journal: Cells

Article Title: Exogenous Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells

doi: 10.3390/cells10010175

Figure Lengend Snippet: Characterization of microsomal fractions (CMF) purified from control and PA- or DHA-treated breast cancer MDA-MB-231 and MCF7 cells (50 µM) for 72 h: the purity of the microsomal fractions (endoplasmic reticulum (ER)) is evaluated using positive ER markers, such as Calnexin, and markers from other cell compartments, such as HSP60 and histone H3.

Article Snippet: The mouse monoclonal anti-HSP60 (66041) antibody was purchased from Proteintech, Deansgate, Manchester, UK.

Techniques: Purification, Control

Journal: Fly

Article Title: secHsp70 as a tool to approach amyloid-β42 and other extracellular amyloids

doi: 10.1080/19336934.2017.1291104

Figure Lengend Snippet: Comparative analysis of heat shock chaperones against intracellular and extracellular amyloids in the Drosophila eye. Panels show fresh eyes and SEM images from flies of the indicated genotypes. (A) Co-expression of intracellular Atx3-Q78 with LacZ results in severe depigmentation and poorly differentiated lenses compared with control flies expressing LacZ alone. However, co-expression of Atx3-Q78 with cytosolic Hsp40 and Hsp70 results in a strong rescue of these phenotypes. Note that cytosolic Hsp27 and Hsp60 do not modify Atx3-Q78 toxicity. (B) Co-expression of Aβ42 with a control LacZ transgene leads to small, glassy eyes with severe ommatidial disorganization compared with control flies expressing LacZ alone. Note that co-expression of Aβ42 with secHsp70 results in bigger and healthier eyes with almost perfect organization of the ommatidial lattice. In contrast, secHsp27, secHsp40 and secHsp60 do not modify the Aβ42-induced phenotype. Eye-specific expression of UAS transgenes was directed with the gmr-Gal4 driver and all UAS constructs were inserted into the same landing site. Insets show a magnification of the ommatidia.

Article Snippet: The cDNAS encoding for human Hsp27 (a gift from H. Kampinga), Hsp40 (Addgene #19468), Hsp60 (Origene SC111640) and Hsp70 (a gift from N. Bonini) were isolated from their respective plasmids and subcloned with and without signal peptide into the injection vector pJFRC-MUH (Addgene #26213).

Techniques: Expressing, Construct