Journal: bioRxiv

Article Title: The Leader of the Capsid protein from Feline calicivirus must be palmitoylated and form oligomers through disulfide bonds for efficient viral replication

doi: 10.1101/2024.06.07.597891

Figure Lengend Snippet: A) CrFK cells were treated with 5, 10, 20, and 50 μM of 16F16, and cell viability was assessed by an MTT assay. B) Total protein extracts from mock-infected or FCV-infected cells at an MOI of 5 for 5 h, untreated or treated with 5, 10, 20, and 50 μM of 16F16 were obtained and levels of LC and VP1 proteins were determined by western blotting. Actin was used as a loading control. C) LC and D) VP1 band intensities from scanned images were quantified using ImageJ software and shown as relative expression. Standard deviations were obtained from three independent experiments. Values of p < 0.000 (***) calculated by T-test using GraphPad Prism 8.0 software are indicated. E) Total protein extracts from FCV-infected cells at an MOI of 5 for 5 h, untreated (0) or treated with 5 and 10 μM of 16F16 were obtained and analyzed by SDS-PAGE in the presence (+) or absence (-) of 5% β-ME. The presence of the LC protein monomers (M) and dimers (D) was determined by western blotting. F) Permeabilized and G) non-permeabilized mock-infected and FCV-infected cells at an MOI of 5 for 5 h and treated with DMSO or 10 μM of 16F16 were immunostained with an anti-LC serum (green) and anti-PDIA3 (red) antibodies. DAPI was used for nuclear (blue) staining. The cells were examined in a Zeiss LSM 700 confocal microscope. Images correspond to a z-stack of 15 slices and represent at least three independent experiments. Merged images are indicated. Colocalization rates were calculated by Pearson’s coefficient correlation using Icy software ( http://icy.biomageanalysis.org ).

Article Snippet: For 16F16 (Santa Cruz Biotechnology), resuspended in DMSO to 0.311M and stored at -80°C, cells were treated similarly.

Techniques: MTT Assay, Infection, Western Blot, Software, Expressing, SDS Page, Staining, Microscopy

Journal: Cells

Article Title: Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro

doi: 10.3390/cells13110906

Figure Lengend Snippet: PDI inhibition affects the expression of HO-1, SUN1, SUN2 and Lamin A/C proteins in MDA-MB-231 cells. ( A ) Representative confocal microscopy images of HO-1 (top row) and PDIA1 (lower row) immunostained MDA-MB-231 cells treated with media only, DMSO (vehicle), 5 μM 16F16, and 2.5 μM PACMA31. Insets (only the red channel is displayed) represent higher magnifications of areas indicated by dashed lines. Arrowheads mark the ER/NE staining of HO-1. Nuclei are visualised by DAPI stain. Scale bar top row: 20 μm; scale bar lower row: 5 μm. ( B ) Quantification of the percentage (%) of cells that exhibit prominent ER/NE HO-1 localisation for MDA-MB-231 cells incubated with media only (M), DMSO (vehicle; D), 5 μM 16F16 (16F) and 2.5 μM PACMA31 (PAC); >150 cells analysed per condition. ( C ) Immunoblot analysis of PDIA1 and HO-1 expression in PDI-inhibited (16F, PAC) and control MDA-MB-231 cells, normalised against GAPDH protein levels, which indicates equal loading of protein. ( D ) Quantification of PDIA1 protein expression in PDI-inhibited (16F, PAC) versus untreated (M, D) MDA-MB-231 cells. ( E ) Quantification of HO-1 protein levels in untreated controls (M, D) versus PDI-inhibited (16F, PAC) MDA-MB-231 cells. ( F ) Representative Western blot analysis of SUN1, SUN2, Lamin A/C, Lamin B1, and GAPDH expression in untreated versus PDI-inhibited cells. ( G – I ) Quantification of SUN1 ( G ), SUN2 ( H ), and Lamin A/C ( I ) protein expression levels. The data in graphs ( B , D , E , G – I ) are represented as the mean ± SEM, with a sample size of n = 3. Statistical significance was conducted using one-way ANOVA with a Dunnett’s post-hoc test relative to the baseline DMSO ( D ) control treatment. Lack of significance is denoted as “ns”, whereas the asterisk (*) indicates p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: After 24 h, the cells were treated with the PDI inhibitors, either PACMA31 or 16F16 (Sigma-Aldrich, Gillingham, UK), at concentrations ranging from 1 to 10 μM, or incubated with their subsequent controls, either media (control) or 0.025% DMSO only (vehicle).

Techniques: Inhibition, Expressing, Confocal Microscopy, Staining, Incubation, Western Blot

Journal: Cells

Article Title: Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro

doi: 10.3390/cells13110906

Figure Lengend Snippet: PDI inhibition disrupts the nuclear envelope localisation of Nesprin-2 isoforms, including Nesprin-2 giant, and results in the formation of perinuclear F-actin rings. ( A , A′ ) Representative immunofluorescence microscopy images of MDA-MB-231 cells incubated with media, DMSO (vehicle), 5 μM 16F16, and 2.5 μM PACMA31, immunostained with Nesprin-2 giant (N2G) isoform-specific (Ab204308) and generic Nesprin-2 isoform (pAbK1) antibodies. DAPI denotes nuclear staining. Insets (only the red channel is displayed) are higher magnifications of dashed boxed areas. Arrowheads indicate the Nesprin-2 presence at the NE. Note the absence of Nesprin-2 staining in the nuclei of PDI-inhibited cells. Scale bar for panel A : 10 μm; scale bar for panel A′ : 20 μm. ( B ) Fluorescence microscopy examination of TRITC-phalloidin (labels F-actin) and DAPI-stained MDA-MB-231 control cells relative to PDI-inhibited cells. Note that the localisation of F-actin in control cells is mainly cortical. Upon PDI inhibition a substantial number of cells (asterisks) exhibit F-actin staining around the NE (arrowheads). Insets (only the red channel is displayed) are higher magnifications of the regions delineated by dashed lines. Scale bar: 20 μm. ( C ) Quantitative examination of the Nesprin-2 Ab204308 antibody (detects Nesprin-2 giant; N2G) and pAbK1 antibody NE staining pattern in control (Media [M], vehicle [D]) and PDI-inhibited (16F16 [16F], PACMA31 [PAC]) cells, with more than 300 cells examined for each condition. ( D ) Quantification of the percentage (%) of cells exhibiting perinuclear F-actin rings under control (M, D), and PDI-inhibited (16F, PAC) conditions is indicated. Over 400 cells were analysed for each specific condition. The data in graphs ( C , D ) are represented as the mean ± SEM, with a sample of n = 3. Statistical significance was determined using one-way ANOVA with a Dunnett’s post-hoc test, comparing to the vehicle treatment (DMSO; [D]). Non-significant differences are denoted as “ns”, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: After 24 h, the cells were treated with the PDI inhibitors, either PACMA31 or 16F16 (Sigma-Aldrich, Gillingham, UK), at concentrations ranging from 1 to 10 μM, or incubated with their subsequent controls, either media (control) or 0.025% DMSO only (vehicle).

Techniques: Inhibition, Immunofluorescence, Microscopy, Incubation, Staining, Fluorescence

Journal: Cells

Article Title: Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro

doi: 10.3390/cells13110906

Figure Lengend Snippet: PDI inhibition affects the Vimentin and Keratin 8/18 intermediate filament cytoskeleton in MDA-MB-231 cells. ( A ) Exemplary immunofluorescence microscopy images of the Vimentin (Vim) and Keratin 8/18 (K8/18) cytoskeletal organisation in control (media [M] and DMSO [D; vehicle]) and 5 μM 16F16 (16F) and 2.5 μM PACMA31 (PAC)-treated cells. The arrowheads indicate the existence of perinuclear cytoskeletal aggregations. Insets (only the red channel is displayed) are higher magnifications of the dashed boxed areas. Asterisks denote cells with a well-dispersed IF cytoskeleton devoid of aggregates. Nuclei are visualised by DAPI staining. Scale bar: 20 μm. ( B ) The percentage (%) of cells displaying perinuclear Vimentin aggregates is depicted in both untreated (M), DMSO (D; vehicle), and drug-treated (16F and PAC) cells. Over 300 cells were examined per condition. ( C ) Shows the percentage (%) of cells exhibiting perinuclear K8/18 aggregates in both untreated and drug-treated cells. More than 300 cells were examined per specific condition. ( D ) Western blot analysis of Vimentin and K8/18 protein expression levels in control and PDI-inhibited cells. Equal protein loading was confirmed using antibodies against GAPDH. ( E – G ) Comparative quantification of Vimentin ( E ), Keratin 8 ( F ), and Keratin 18 ( G ) protein expression levels (fold change) in untreated (M) and drug-treated cells in relation to the normalised vehicle ( D ) control sample.The data in graphs ( B , C , E – G ) are represented as the mean ± SEM, with a sample of n = 3. Statistical significance was determined using one-way ANOVA with a Dunnett’s post-hoc test, compared to the vehicle treatment (DMSO; [D]). Non-significant differences are denoted as “ns”, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: After 24 h, the cells were treated with the PDI inhibitors, either PACMA31 or 16F16 (Sigma-Aldrich, Gillingham, UK), at concentrations ranging from 1 to 10 μM, or incubated with their subsequent controls, either media (control) or 0.025% DMSO only (vehicle).

Techniques: Inhibition, Immunofluorescence, Microscopy, Staining, Western Blot, Expressing

Journal: Cells

Article Title: Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro

doi: 10.3390/cells13110906

Figure Lengend Snippet: Pharmacological inhibition of PDI activity in MDA-MB-231 cells reduces cell proliferation, 2D cell migration, and cell polarity, while promoting their migration through space-restrictive 5 μm porous surfaces. (A) Confocal microscopy micrographs of EdU proliferation assay results depict untreated, vehicle-treated (DMSO), and drug-treated (i.e., 5 μM 16F16 and 2.5 μM PACMA31) MDA-MB-231 cells. DAPI staining denotes nuclei. Scale bar: 50 μm. ( B ) Comparative quantification of EdU incorporation (fold change) in untreated (M) and drug-treated (5 μM 16F16 [16F] and 2.5 μM PACMA31 [PAC]) cells in relation to the normalised vehicle (D) controls. ( C ) Phase contrast images of cell wounding experiments at 0 h and 24 h post-wounding, involving untreated and PDI-inhibited cells. Scale bar: 100 μm. ( D ) Graph illustrating the percentage (%) of wound closure at 0 h, 6 h, 12 h, and 24 h time points. ( E ) Confocal microscopy image of the free wound edge of a MDA-MB-231 2D monolayer stained for the Golgi apparatus (GM-130) and the nucleus (DAPI). The direction of cell movement is indicated with the white arrow. Scale bar: 10 μm. The schematic on the right illustrates the criteria employed to distinguish between polarised and non-polarised cells. Cells were considered polarised when the Golgi organelle (depicted in green) was oriented within a 180-degree radius (depicted in grey) facing the free wound edge. The nucleus is depicted as a blue circle. ( F ) The graph illustrates the percentage (%) of polarised cells for the specified drug-treatments relative to the control conditions. ( G ) A schematic representation of cell migration is shown, employing inserts containing a space-restrictive porous (Diameter: 5 μm) membrane (dashed purple line) and serum-based attractants. The light pink shading indicates the lack of serum, while the dark pink shading denotes the presence of serum. The schematic captures cell behaviour at the 0 h (0 h) seeding and 24 h (24 h) post-seeding time points. ( H ) Graph indicating the absolute numbers of cells that migrated to the basal side of the membranous porous insert under the specified concentrations of the 16F16 drug, in comparison to control conditions. ( I ) The presented data illustrate the absolute numbers of cells that migrated to the basal side of the porous membrane insert under the defined concentrations of the PACMA31 drug, relative to control conditions. The data in graphs ( B , D , F , H , I ) are represented as the mean ± SEM, with a sample of n = 3. Statistical significance was determined using one-way ANOVA with a Dunnett’s post-hoc test, compared to the vehicle treatment (DMSO; [D]). Non-significant differences are denoted as “ns”, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: After 24 h, the cells were treated with the PDI inhibitors, either PACMA31 or 16F16 (Sigma-Aldrich, Gillingham, UK), at concentrations ranging from 1 to 10 μM, or incubated with their subsequent controls, either media (control) or 0.025% DMSO only (vehicle).

Techniques: Inhibition, Activity Assay, Migration, Confocal Microscopy, Proliferation Assay, Staining, Membrane, Comparison

Journal: Cells

Article Title: Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro

doi: 10.3390/cells13110906

Figure Lengend Snippet: A schematic depiction of SUN2 macromolecular organisation in the nuclei of untreated MCF10A and MDA-MB-231 cells ( A ), along with the consequences of PDI inhibition on LINC complex composition and perinuclear F-actin structure ( B ). ( A ) Nuclear 2D projection (XY plane). SUN2 macromolecular assemblies are depicted as small coloured circles. The colours create a gradient with varying shades from “light yellow” to “dark green”, aiming to reflect changes in pixel intensity, transitioning from lower to higher values. Note the prevalence of “green” SUN2 structures at the periphery and “light yellow” SUN2 complexes in the centre; aiming to reflect that in MCF10A nuclei, SUN2 staining pixel intensity is at the highest at the periphery and lowest in the centre. The distribution of SUN2 complexes is even throughout the entire nucleus. In MDA-MB-231 cells, the gradient of SUN2 pixel intensity from the periphery to the centre is maintained, yet the organisation of SUN2 complexes is less orderly. The insets below aim to offer a more detailed view at the molecular level. For simplicity, SUN2 is depicted as a trimer, despite ample evidence that other molecular assemblies exist. The SUN2 cysteines are depicted as small “lollipops”. Reduced cysteines (SH) are illustrated in yellow, while oxidised cysteines (S–S) are portrayed in orange. Within MCF10A nuclei, oxidised SUN2 trimers are present, displaying a preference for localisation at the nuclear periphery. This localisation could potentially facilitate uniform SUN2 complex spacing and limit the overcrowding of SUN2 molecules. In stark contrast, MDA-MB-231 cells exhibit SUN2 trimers lacking stabilisation and equal spacing through disulphide bonds, resulting in SUN2 aggregation, as indicated by the “dark green” colouration of SUN2 complexes. ( B ) The primary consequences on an MDA-MB-231 cell following PDI inhibition are summarised, with a specific focus on the LINC complex and F-actin architecture. Protein expression effects common to both PACMA31 and 16F16 treatments are shown in bold (i.e., Lamin A/C and Keratin 18 downregulation). Upon PDI inhibition, the expression of anchoring proteins for Nesprin-2, such as Lamin A/C and SUN-domain proteins, are reduced. This potentially leads to the untethering of Nesprin-2 from the outer nuclear membrane. The model suggests that the combination of these molecular events results in decreased nuclear stiffness, which facilitates cellular migration through space restrictive scaffolds in vitro. Moreover, it is hypothesised that the effects on nuclear pliability and structure are counteracted by the development of an F-actin perinuclear cage in drug-treated cells.

Article Snippet: After 24 h, the cells were treated with the PDI inhibitors, either PACMA31 or 16F16 (Sigma-Aldrich, Gillingham, UK), at concentrations ranging from 1 to 10 μM, or incubated with their subsequent controls, either media (control) or 0.025% DMSO only (vehicle).

Techniques: Inhibition, Staining, Expressing, Membrane, Migration, In Vitro