Journal: Pharmaceutics

Article Title: Repurposing of Ciclopirox to Overcome the Limitations of Zidovudine (Azidothymidine) against Multidrug-Resistant Gram-Negative Bacteria

doi: 10.3390/pharmaceutics14030552

Figure Lengend Snippet: Minimum inhibitory concentrations (MICs) of test antibiotics against Gram-negative bacterial strains.

Article Snippet: As shown in b, strongly AZT-resistant E. coli from ATCC 25922 cells (here named AZT-R E. coli ) were generated within 2 d with an AZT MIC >1000-fold higher than that of control cells and >20-fold higher than that of Keio- tdk ( ; ).

Techniques: Laser Capture Microdissection

Journal: Pharmaceutics

Article Title: Repurposing of Ciclopirox to Overcome the Limitations of Zidovudine (Azidothymidine) against Multidrug-Resistant Gram-Negative Bacteria

doi: 10.3390/pharmaceutics14030552

Figure Lengend Snippet: Confirmation of Mcr-1 expression in E. coli ATCC 25922. ( a ) Confirmation of mcr-1 expression from the related plasmid. PCR amplification using pQE-60-specific primer sets was performed for KS7001 (pQE-60, lane 1) and KS8001 (pQE-60- mcr-1 , lane 2), which overproduced a His-tagged Mcr-1 protein , on 1% agarose gel wis shown. M and asterisk (*) indicate the DNA size marker (GeneRuler 1kb Plus DNA ladder; Thermo Scientific, MA, USA) and a non-specific PCR product, respectively. ( b ) Detection of Mcr-1 protein. Mcr-1 protein from KS8001 was detected by Western blotting with an Anti-His tag Antibody (Sino Biological, Wayne, PA, USA). Image acquisition and quantitative analysis were performed by using ChemiDoc TM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab TM Software (ver 5.2.1; Bio-Rad, Hercules, CA, USA).

Article Snippet: As shown in b, strongly AZT-resistant E. coli from ATCC 25922 cells (here named AZT-R E. coli ) were generated within 2 d with an AZT MIC >1000-fold higher than that of control cells and >20-fold higher than that of Keio- tdk ( ; ).

Techniques: Expressing, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Marker, Western Blot, Imaging, Software

Journal: Pharmaceutics

Article Title: Repurposing of Ciclopirox to Overcome the Limitations of Zidovudine (Azidothymidine) against Multidrug-Resistant Gram-Negative Bacteria

doi: 10.3390/pharmaceutics14030552

Figure Lengend Snippet: Generation of resistance phenotypes by AZT or CPX. ( a ) Schematic representation of the resistance generation method. Bactericidal activity of ( b ) AZT or ( c ) CPX against E. coli ATCC 25922. Representative data from n = 3 experiments, as described in are shown. Relative increase of resistance of ATCC 25922 to ( d ) AZT or ( e ) CPX. The relative increase of resistance as a fold change with respect to non-drug-treated ATCC 25922 or Keio- tdk cells (set to 1 for each set) was calculated based on MIC changes from ( b , c ). The values shown in the graph at the indicated days are averaged values from n = 3 experiments, with standard deviations ( p < 0.05).

Article Snippet: As shown in b, strongly AZT-resistant E. coli from ATCC 25922 cells (here named AZT-R E. coli ) were generated within 2 d with an AZT MIC >1000-fold higher than that of control cells and >20-fold higher than that of Keio- tdk ( ; ).

Techniques: Activity Assay

Journal: Pharmaceutics

Article Title: Repurposing of Ciclopirox to Overcome the Limitations of Zidovudine (Azidothymidine) against Multidrug-Resistant Gram-Negative Bacteria

doi: 10.3390/pharmaceutics14030552

Figure Lengend Snippet: Bactericidal activity of CPX in AZT resistant strains. ( a ) PCR amplification of the tdk -encoding gene. Sequence information for ATCC 25922 was obtained from the NCBI database (accession No. CP009072.1). Sequence alignment of ( b ) the tdk gene. DNA sequencing results of tdk -encoding region from AZT-R and ATCC 25922, read using the primers Keio- tdk -F and -R, were aligned using Clustal Omega (ClustalW2, v2.1, http://www.clustal.org ; accessed on 10 January 2022) . ( c ) Bactericidal activity of CPX. The bactericidal activity of CPX in AZT-resistant E. coli cells (AZT-R or AZT- tdk -R) and control cells (ATCC 25922) was determined at the indicated concentrations. Data shown here are from one representative experiment from triplicate experiments. The LB agar plates were imaged with a digital camera (Samsung NX200, Suwon, Korea).

Article Snippet: As shown in b, strongly AZT-resistant E. coli from ATCC 25922 cells (here named AZT-R E. coli ) were generated within 2 d with an AZT MIC >1000-fold higher than that of control cells and >20-fold higher than that of Keio- tdk ( ; ).

Techniques: Activity Assay, Amplification, Sequencing, DNA Sequencing

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RNAi screening identifies RABGAP1L as an IAV restriction factor (A) Schematic representation of recombinant IAV WSN/33 in which the coding region for the hemagglutinin (HA) glycoprotein has been replaced by Renilla luciferase (WSN/33- Renilla ). (B) RNAi-screening experimental workflow. (C) MRC-5-HA cells were transfected for 30 h with individual siRNAs targeting MX1 or IFITM3 or with a non-targeting (NT) control siRNA. Following stimulation with IFNα2 (1,000 U/mL or mock) for 16 h, cells were infected with WSN/33- Renilla (MOI 5 PFU/cell) in the presence of the live-cell substrate EnduRen. Luciferase activity was monitored up to 12 h post-infection (p.i.), and the area under the curve (AUC) was calculated as indicated. Mean values from 50 technical replicates across two independent biological experiments are plotted, with error bars representing SDs. (D) Hit criteria for RNAi screening. In a primary screen following the workflow in (B), 100 putative ISGs were silenced with four individual siRNAs each. Twenty-two genes met the threshold, and 20 were re-tested in a confirmation screen. Applying the same hit criteria, a total of 8 putative ISGs were confirmed in both screening rounds. (E) Heatmap showing Z scores of positive controls ( MX1 and IFITM3 ) and the top 8 hits from the two RNAi-screening rounds. Columns represent individual siRNAs targeting genes listed in rows. See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Luciferase, Transfection, Infection, Activity Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: IFN-mediated restriction of IAV by RABGAP1L (A) A549 cells were transfected with the indicated siRNAs for 32 or 60 h prior to lysis and assessment of cell viability using CellTiter-Glo. An NT siRNA and an siRNA targeting IRF9 were used as negative controls. siRPS is an siRNA targeting the essential gene RPS27A and thus acted as a positive control for cell toxicity. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (B and C) A549 cells were transfected with the indicated siRNAs 30 h prior to IFNα2 treatment (1,000 U/mL or mock). Sixteen hours post-IFN stimulation, cells were infected with WSN/33- Renilla (MOI 1 PFU/cell), and luciferase activity was monitored every 2 h for a total of 12 h. The NT siRNA and siRNA targeting IRF9 were used as controls. (C) The AUC was calculated from measured relative light units (RLUs) over time. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (D) In parallel to (B) and (C), cells were harvested for western blot analysis 16 h post-IFN stimulation. Proteins of interest were detected as indicated. RABGAP1L (RG1L) isoforms corresponding to detected bands are highlighted. (E) Schematic representation of RABGAP1L isoforms A, G, H, and I, showing the phosphotyrosine-binding (PTB) domain, the kinesin-like (kin) domain, and the Tre-2/Bub2/Cdc16 (TBC) domain. Isoform G further contains a domain of unknown function (DUF3084). (F) Immunofluorescence analysis of A549 cells stably expressing either empty vector (EV) or RABGAP1L isoforms A, G, H, and I. Cells were fixed and stained for RABGAP1L (red); nuclei were stained with DAPI (blue). Scale bar represents 25 μm. Representative confocal-microscopy images from at least two biologically independent experiments are shown. (G) Cells described in (F) were harvested for western-blot analysis. Proteins of interest were detected with the indicated antibodies. Images are representative of three biologically independent experiments. (H) Cells described in (F) and (G) were treated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected 48 h p.i. and titrated on Madin-Darby canine kidney (MDCK) cells to determine viral titers. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance in (C) and (H) was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001; ns, non-significant). See also and .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Transfection, Lysis, Positive Control, Infection, Luciferase, Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L overexpression restricts selected positive- and negative-sense RNA viruses (A) A549 cells stably expressing GFP or RABGAP1L (RG1L) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with different Renilla luciferase-encoding IAVs: H1N1 (WSN/33, MOI 1 PFU/cell), pdmH1N1 (Neth/09, MOI 5 PFU/cell), or H5N1 (Viet/04, MOI 0.5 PFU/cell). EnduRen live-cell substrate was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs up to 11 h p.i. (B) Huh-7 cells stably expressing GFP or RG1L were treated as described in (A) and infected with WSN/33- Renilla (MOI 1 PFU/cell) or HCoV-229E- Renilla (MOI 5 PFU/cell). EnduRen was supplemented, and the luciferase activity was measured every 2 h for a total of 11 h. RLUs were used to calculate the AUC. (C–E) A549 cells expressing EV or RG1L were stimulated with IFNα2 (10, 100 or 1,000 U/mL or mock) for 4 h prior to infection with VSV-GFP (MOI 1 PFU/cell) (C) or for 16 h prior to infection with SeV-GFP (MOI ∼1 PFU/cell) (D) and NDV-GFP (MOI 1 PFU/cell) (E). GFP intensity was measured every 2 h for up to 72 h. The AUC was calculated from total green integrated intensity. (F and H) Calu-3 (F) or Vero-CCL81 (H) cells stably expressing GFP or RG1L were treated with IFNα2 (10, 100, or 1,000 U/mL or mock) for 16 h, followed by infection with WSN/33- Renilla (MOI 1 PFU/cell). EnduRen was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs. (G and I) Calu-3 (G) or Vero-CCL81 (I) cells stably expressing GFP or RG1L were treated as described in (F) prior to infection with SARS-CoV-2 (MOI 0.1 PFU/cell). Supernatants were collected 24 h p.i., and viral titers were determined by plaque assay in Vero-E6 cells. (A–I) Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined comparing GFP-overexpressing with RG1L-overexpressing cells in equal treatment conditions in all panels using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.0002, ∗∗∗∗ p < 0.0001; ns, non-significant).

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Over Expression, Stable Transfection, Expressing, Infection, Luciferase, Activity Assay, Plaque Assay, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: The antiviral function of RABGAP1L relies on its catalytically active TBC domain and residues implicated in endosomal trafficking (A) Schematic representation of RG1L WT and the 421 mutant (RG1L 421) which lacks the C-terminal region downstream of the kin domain. (B) Immunofluorescence analysis of A549 cells stably expressing EV, RG1L WT, or RG1L 421. Cells were fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (C) A549 cells stably expressing GFP, RG1L WT, or RG1L 421 were stimulated with IFNα2 (1,000 U/mL or mock) for 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected after 48 h and titrated on MDCK cells. (D) Schematic representation of the TBC domain of RABGAP1L and the localization of mutants R584A (R mut ), Q621A (Q mut ), R584A-Q621A (RQ mut ), and KK784EE (KK mut ). KK mut has previously been shown to prevent interaction with the AnkB death domain (DD). (E) Western blot validation of RABGAP1L expression in A549 cells stably expressing RG1L WT or the indicated mutants. (F) Immunofluorescence analysis of cells described in (E) (here, EV was used as a control), fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (G) Cells described in (E) were infected with WSN/33- Renilla (MOI 1 PFU/cell) following treatment with IFNα2 (1,000 U/mL or mock) for 16 h. The AUC was calculated from RLU values taken up to 11 h p.i. For (B), (E), and (F), representative data from three biologically independent experiments are shown. For (C) and (G), mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001). See also Figure S3 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Mutagenesis, Immunofluorescence, Stable Transfection, Expressing, Staining, Infection, Western Blot, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: Proximity-labeling-based proteomics identifies the RABGAP1L host interactome (A) Schematic representation of TurboID-V5-tagged (T-V5) GFP (negative control) carrying a nuclear-export sequence (NES) or T-V5-tagged RABGAP1L (T-V5-RG1L). (B) Constructs described in (A) were stably expressed in A549 cells, and their expression was validated by immunofluorescence using an α-V5 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (C) Western blot analysis of cells described in (B) compared with A549 cells stably expressing untagged GFP or RABGAP1L (RG1L). Proteins of interest were detected with the indicated antibodies. (D) Cells described in (C) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33- Renilla (MOI 1 PFU/cell). The AUC was calculated from RLU values taken up to 11 h p.i. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (E) Workflow of the TurboID proximity-labeling approach. Cells described in (B) were treated with IFNα2 (1,000 U/mL or mock) for 16 h, followed by treatment with biotin (500 μM) for 15 min. Following streptavidin-based affinity purification, peptides were generated and subjected to mass-spectrometry analyses. (F) Interactors specific to RABGAP1L (as compared to GFP-NES) identified using the protocol described in (E). Hits are listed with their gene names and sorted according to previously described functions. Most hits were identified in non-IFNα2-treated samples. Hits marked with an asterisk ( ∗ ) were identified in the presence and absence of IFNα2, and hits marked in bold were only identified in IFNα2-treated samples. (G) A549 cells stably expressing constructs introduced in (A) or T-V5-tagged RABGAP1L KK mut and RQ mut were subjected to the proximity labeling approach outlined in (E). Following streptavidin-based affinity purification (samples termed “eluates”), total lysates and eluates were analyzed by western blot. Proteins were detected with the indicated antibodies. Data obtained in (B), (C), and (G) are representative of three biologically independent experiments. For (D), statistical significance was determined using one-way ANOVA following log transformation (ns, non-significant). See also and Figure S4 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Labeling, Negative Control, Sequencing, Construct, Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot, Infection, Affinity Purification, Generated, Mass Spectrometry, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L expression impacts host endosomal function and IAV uptake (A–C) A549 cells stably expressing RABGAP1L WT, the 421-truncation mutant, or EV were infected with WSN/33 (MOI 5 PFU/cell) for 1 h on ice. Three hours after incubation at 37°C, cells were fixed and stained with antibodies against RABGAP1L (red) and NP (green) (A). Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (B and C) Green mean fluorescent intensities (MFIs) of nuclear NP signals were quantified from fluorescent-microscopy images from (A) using ImageJ software. Individual cells are represented by single dots (B). Mean values of data from three biologically independent experiments in (B), normalized to EV, are shown in (C). (D) MDCK cells, expressing the constructs described in (A), were infected for 4 h at 37°C with WSN/33-pseudotyped β-lactamase-matrix protein (BlaM1) fusion protein VLPs prior to quantification of entry-positive cells via flow cytometry. Data represent means, with error bars showing SDs, from three biologically independent experiments. Individual data points are shown. (E) Experimental setup for immunofluorescence-based confocal microscopy to track early stages during IAV entry. Following infection with WSN/33 (MOI 25 PFU/cell or mock) for 1 h at 4°C, cells were fixed at the indicated timepoints. (F) A549 cells stably expressing RABGAP1L (RG1L) or EV were subjected to the experimental setup described in (E). The MFI of HA signals (green) at 0 min p.i. were quantified from confocal-microscopy images shown in Figure S6 A using ImageJ. Individual cells are represented by single dots. (G) Quantification of co-localizations between EEA1 and HA from confocal images shown in (H) and Figure S6 A using Imaris. Individual cells are represented by single dots. (H) Immunofluorescence analysis of RABGAP1L or EV-expressing A549 cells treated as described in (E). Cells were stained for early endosomes (EEA1, magenta), viral proteins (HA, green), and nuclei (DAPI, blue). Scale bar represents 25 μm. White arrows indicate co-localizations between EEA1 and HA. Representative images of at least nine analyzed cells per time point from at least two biologically independent experiments. (I) Quantification of co-localizations between EEA1 and HA from confocal images shown in Figure S6 B. Individual cells are represented by single dots. (J and K) Cells described in (A) were serum starved for 2 h prior to treatment with Dynasore (Dyn.; 100 μm) or DMSO for 1 h at 37°C. Cells were then incubated with Alexa-Fluor-488-conjugated transferrin (Tf-488) for 1 h at 4°C followed by a 10-min incubation at 37°C prior to fixation. (J) MFI quantification of Tf-488 signals from confocal-microscopy images shown in (K) using ImageJ software. Individual cells are represented by single dots. (K) Cells were stained with anti-transferrin receptor (TfR) antibody (magenta) and DAPI (blue) prior to analysis by confocal microscopy. Scale bar represents 25 μm. Representative images of at least 25 analyzed cells from two biologically independent experiments. Statistical significance was determined using unpaired nonparametric t test (B, F, G, and J), unpaired one-way ANOVA (C and D), or ordinary two-way ANOVA (I) ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, non-significant). See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Expressing, Stable Transfection, Mutagenesis, Infection, Incubation, Staining, Microscopy, Software, Construct, Flow Cytometry, Immunofluorescence, Confocal Microscopy

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Cell Viability Assay, Mutagenesis, Clone Assay, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: (A) Experimental strategy to delete Smad2 and Smad3 in ECs. Red arrows indicate TAM injections. (B and C) Western blot for SMAD2 or SMAD3 and β-actin using ECs isolated from lungs of WT ( Smad2l/l or Smad3l/l ) and Smad2iEKO and Smad3iEKO mice, respectively. (D) IsoB4/α-SMA double staining of P12 retinas of the indicated genotypes. (E) Quantification of vascular parameters of Smad2/3l/l and Smad2/3iEKO P12 retinas (mean ± SEM, each dot represents one retina, *p < 0.05, **p < 0.01, Mann-Whitney U test). (F) Experimental strategy to delete Smad4 in ECs. Red arrows indicate TAM injections. (G) IsoB4 and α-SMA double staining of P12 Smad4l/l and Smad4iEKO retinas. (H) Quantification of vascular parameters of P12 retinas of the indicated genotypes (mean ± SEM, each dot represents one retina, *p < 0.05, **p < 0.01, **p < 0.001, Mann-Whitney U test). (I) Double immunostainings for IsoB4 and APOE, VWF, TXNDR1, or PLVAP of retina flat mounts of the indicated genotypes at P12. A, artery; V, vein.

Article Snippet: Rabbit Anti-Smad3 (C67H9) monoclonal antibody , Cell Signaling , Cat#9523; RRID: AB_2193182.

Techniques: Western Blot, Isolation, Double Staining, MANN-WHITNEY

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-Smad3 (C67H9) monoclonal antibody , Cell Signaling , Cat#9523; RRID: AB_2193182.

Techniques: Recombinant, Saline, Fluorescence, Plasmid Preparation, Modification, Western Blot, cDNA Synthesis, SYBR Green Assay, Imaging, RNAscope, Multiplex Assay, Gene Expression, Software, Laser-Scanning Microscopy

Journal: Cell reports

Article Title: APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release

doi: 10.1016/j.celrep.2023.113252

Figure Lengend Snippet:

Article Snippet: Donkey anti-mouse Biotin-SP , Jackson Immuno Research Labs , Cat#: 715-065-150; RRID: AB_2307438.

Techniques: Recombinant, Protease Inhibitor, Blocking Assay, Plasmid Preparation, Stripping Membranes, Electron Microscopy, Saline, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Extraction, Software, Generated, Microscopy, Laser-Scanning Microscopy, Imaging, Spectrophotometry

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Intracellular P. gingivalis ( P. g ) Significantly Induces and Co-Localizes with LC3C, an Isomer of LC3, and this Specific Event is Highly Dependent on HSp27 for Successful Autophagic Survival. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated for 6 or 24 h. ( A ) GECs were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Following HSp27 depletion, P. g appeared to readily start to degrade. Representative transmission electron microscopy images of P. g -infected GECs were also taken at 80 kV and 100000x magnification. Scale bar is 800 nm. ( B ) 6 h and 24h P. g-infected GECs were also stained for P. g (rabbit anti-P . g ; Alexa 488; green) and LC3C (mouse anti-LC3C; Alexa 568; red) to examine whether LC3C characterizes P. g -specific autophagosomes. These cells were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. The range of z-stacks was kept consistent and representative images were selected from the mid-ranged sections. ( Bi ) The Imaris software was utilized to obtain a xoomed 63x Orthogonal Image of 24 h P. g infection and found heightened co-localization between P. g and LC3C. LC3C was found to readily colocalize with P. g, having an average Pearson correlation coefficient of 0.96 via the Imaris post-processing software. ( C ) Lysates of infected and HSp27-depleted GECs were also analyzed via western blotting. Non-target controls were performed and not shown. (Ci) Quantitative ImageJ analysis was performed for the western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. **p<.005.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Labeling, Transmission Assay, Electron Microscopy, Infection, Staining, Confocal Microscopy, Software, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Autophagic Lifestyle of P. gingivalis (P. g) is Highly Characterized by Only the LC3C Isoform of LC3, Which is not Increased During Starvation-Induced Autophagy in GECs. (A ) The LC3 A/B lipidation results of the same assay provided in . (B ) GECs were separately treated with LC3B siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6 h. Intracellular P. g survival after LC3B siRNA depletion was determined using a standard antibiotic protection assay using P. g- specific 16S rRNA primers. ( C ) GECs were subjected to starvation conditions in HBSS for 24 h. GECs were then collected and fixed so that immunofluorescence could be performed. GECs were stained for LC3C (rabbit anti-LC3C;Alexa 568; red). GECs were then imaged via confocal microscopy (Super Resolution Zeiss Airyscan LSM 880) at 63x. Western blotting (Not Shown) was utilized to confirm the lack of induction of LC3C I and LC3C II. Data is represented as Mean±SD; n=3; p<0.05 is considered statistically significant (Student two-tailed T-test).

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 Presence Permits the Prolonged Existence of LC3C-characterized, P. gingivalis Specific Autophagosomes by Hampering Canonical Autolysosomal Fusion in Primary GECs. ( A ) Human primary GECs were transfected with mCherry-eGFP-LC3C for 48 h. Select GECs were also treated with 1 uM of the autophagolysosomal fusion inhibitor Bafilomycin A1, 1 uM Pepstatin A, or 5 mM 3-MA. Others were treated with Hsp27 siRNA (100nM) for 24 h. P. g was added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs were then stained for P. g (mouse anti- P.g; Alexa 405; blue) and were mounted. GECs were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. ( Ai ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the high co-localization levels between P. g and the LC3C Reporter System. P. g localized readily to the LC3C construct, with a Pearson correlation coefficient of 0.82. (B) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Scale bar is 40 µm for 63x Magnification. ( Bi ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the co-localization levels between P. g and LAMP-1 in infected and treated GECs. While P. g infected GECs did not exhibit high co-localization with LAMP-1 (Pearson correlation coefficient of .25), their HSp27-depleted counterparts did, with an average Pearson correlation coefficient of 0.83. The Scale bar is 20 µm for 63x Magnification. ( C ) Finally, GECs treated with autophagic inhibitors were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Following HSp27 depletion, P. g appeared to readily start to degrade, however treatment with late-stage autophagic inhibitors Bafilomycin A1 or Pepstatin A appeared to rescue P. g from degradation. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Scale bar is 800 nm.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Construct, Labeling, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Depletion of LC3C via siRNA Collapses P. gingivalis (P. g) -Induced Non-Canonical Autophagosomal Integrity. Human primary GECs were treated with LC3C siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6, 12, or 24 h. ( A ) Intracellular P. g survival after LC3C siRNA depletion was determined using a standard antibiotic protection assay. In brief, any extracellular bacteria were killed via 1h gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular levels of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005. ( B ) P. g -specific autophagosomes were also selectively isolated. Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal images of P. g -specific autophagosomes at 6 h post-infection (63x) were taken utilizing the Super Resolution Zeiss Airyscan LSM 880.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Bacteria, Synthesized, Two Tailed Test, Isolation, Staining, Infection

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Causes the Nucleation of Hsp27-Mediated LC3C Accumulation and Lipidation; this Specific Assembly is Highly Dependent on Host Cells’ Redox Potential Determined by eATP Treatments. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated 6 and 24 h. Non-Depleted and HSp27-depleted GECs also were treated with the physiologically-relevant oxidative stress inducer eATP (3mM) treatment for 30 min prior to infection, and were analyzed by western blot.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Infection, Western Blot

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis ( P. g ) Induces and Prolongs the Autophagosomal LC3C/Beclin 1/ATG14 Nucleation Complex in a Manner Dependent upon HSp27 and the Reduced Redox State of Infected GECs as Determined by Isolated P. g- Specific Autophagosomes. GECs were treated with HSP27siRNA (100nM) for 48 h. Select GECs were also treated with N-acetyl Cysteine (NAC)(50 uM) for 1 h and/or eATP (3mM) for 30 min. P. g was added at MOI 100 to GECs, which were incubated 6 and 12 h. Autophagosomes were then isolated and prepared for analysis. ( A ) The glutathione (GSH) levels of primary GECs were also measured using chemiluminescence detection. ( B) Isolated autophagosomes were analyzed via western blot. ( Bi ), ( Bii ), ( Biii ) Quantitative ImageJ analysis was performed of each of the western blot results. Data is represented as Mean±SD, where n=3 for results. p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Infection, Isolation, Incubation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 and LC3C Recruit Beclin 1 and ATG14 to Form a Temporal Pro-bacterial Autophagic Complex, which Can Be Disrupted by Increased Oxidative Stress. Human Primary GECs were treated with HSp27siRNA (100nM) for 48 h. Select GECs were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h and/or eATP (3mM) for 30 min. P. gingivalis (P. g) was added at MOI 100 to GECs, which were incubated 6 and 12 h. GECs were then lysed and the extracts were incubated in rabbit anti-LC3C antibody over-night. Samples underwent co-immunoprecipitation. ( A ) The eluted protein complexes were then analyzed by western blot. ( Ai ), ( Aii ), and ( Aiii ) Quantitative ImageJ analysis of western blot results was performed for each of the proteins in question. ( B ) GECs also underwent staining for HSp27 (goat anti-HSp27; Alexa 405; blue), LC3C (rabbit anti-LC3C; Alexa 488; green), and Beclin 1 (sheep anti-Beclin 1; Alexa 568; red), and ATG14 (mouse anti-ATG14; Alexa 647; magenta) to examine the formation of the pro-bacterial autophagic initiation complex. GECs were then imaged via Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. The Imaris software was used to obtain zoomed orthogonal views of ( Bi ) An infected GECs and ( Bii ) a theoretical autophagosome with HSp27, LC3C, ATG14, and Beclin 1 highly co-localized about it. The scale bar is 20 µm for all Magnification. All of the markers were found to have a Pearson correlation coefficient greater than .9 with each other via Imaris, denoting their close theorized interactions. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Immunoprecipitation, Western Blot, Staining, Software, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 and LC3C Selectively and Specifically Partner with One Another to Promote P. gingivalis ( P. g )-Induced Autophagy. P. g was added at MOI 100 to Human Primary GECs, which were incubated 6 and 24 h. ( A ) GECs were stained for LC3C (rabbit anti-LC3C; Alexa 488; green) and HSp27 (mouse anti-HSp37; Alexa 568; red) following infection. HSp27 was found to readily and temporally colocalize with LC3C, having a Pearsons correlation coefficient of .85 at 24 h post infection via the Imaris post-processing software. ( B ) To assess if full length HSp27 is truly capable of binding to full-length LC3C, a far western approach was implemented by probing 5 µg of recombinant LC3C with 10 µg of recombinant HSp27 for one hour. Antibody specificity was accounted for via probing the LC3C blot with monoclonal mouse anti-HSp27 antibody (Not shown), which showed no cross-reactivity.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Staining, Infection, Software, Binding Assay, Western Blot, Recombinant

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 does not Interact with either LC3A or LC3B isoforms in the way that it interacts with LC3C. A Far Western approach was implemented. rLC3A or rLC3B were loaded and incubated with 10 ug of rHSp27. Interactions for ( Bi ) LC3A and ( Bii ) LC3B were then detected by probing the rLC3A or rLC3B blot with mouse Anti-Hsp27 antibody.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Western Blot, Incubation

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Phosphorylated HSp27 (P-HSp27) Preferentially Binds to the C-terminal Tail of LC3C, Inhibiting the Canonical Cleavage of LC3C and Halting the Canonical Maturation of LC3C-Specific Autophagosomes. The structural models of monomeric full-length wild-type ( A ) HSp27 (Uniprot: P04792) and ( B ) LC3C (Uniprot: Q9BXW4) were acquired from the AlphaFold database. Optimized complex configurations between ( C ) LC3C and unmodified HSp27 and ( D ) LC3C and P-HSp27 were then obtained, and the modifications in the theorized interaction sites in their N-terminal regions were highlighted. ( E ) Surface electrostatic potentials of the complexes were additionally mapped, contrasting the varied potentials between the two complexes. ( F ) Finally, the buried surface areas and interaction areas between HSp27 or P-HSp27 and LC3C proteins were also assessed and contact maps were generated.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Generated

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Secretes its Ndk Effector Molecule to Activate HSp27 and Induce Temporal HSp27-LC3C Partnering to Inhibit Canonical LC3C Cleavage by ATG4B and Halt Autolyosomal Fusion in GECs. A) Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h or were transfected with 1 µg of the constitutively activated pFLAG-CMV2-HSP27-S78D/S82D construct for 48h. Select GECs were then jointly treated with the late stage autophagy inhibitors 1 µM Pepstatin A, or 1 µM lactostatin for 24h. Wild-type P. g or ΔNDK P. g was then added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs also underwent staining for HSp27 (goat anti-HSp27; Alexa 405; blue), LC3C (rabbit anti-LC3C; Alexa 488; green), and Beclin 1 (sheep anti-Beclin 1; Alexa 568; red), and ATG14 (mouse anti-ATG14; Alexa 647; magenta) to examine the formation of the pro-bacterial autophagic initiation complex. GECs were then imaged via Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( B ) A diagram was created detailing how LC3C preferentially partners to P-HSp27 over its non-phosphorylated counterpart, causing a confirmational shift to the C-terminal tail of LC3C. This shift results in the inhibition of the final lipidated LC3C tail cleavage by the ATG4B protease, lending to LC3C not disassociating from the autophagosome and halting fusion with the lysosome. ( C ) A diagram was also created to highlight the temporal relationship between HSp27 and LC3C, where LC3C can initially bind to HSp27 but via the actions of Ndk, it preferentially binds to P-HSp27, resulting in a limiting of mature, cleaved LC3C.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Transfection, Construct, Incubation, Staining, Inhibition

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Cross-Sectional Human in Situ Sample and Expression Analyses Support High Levels and Increased Co-localization of P. gingivalis (P. g) , HSp27, and LC3C in Periodontitis-Afflicted Oral Tissues. Publicly available mRNA expression data (GEO accession: GSE79705) was obtained from previously collected and examined periodontitis-afflicted and healthy gingival tissues. This microarray expression data was then analyzed via GEO2R and the relative levels of ( A ) HSp27 and ( B ) LC3C were obtained and compared. Data is represented as Mean±SD, where n=12 and p<0.05 was considered as statistically significant via One-Way Anova. *p<0.05. Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis-afflicted patients were also taken and examined. DAPI staining was utilized to visualize cellular DNA. ( C ) P. g (mouse anti-P . gingivalis ; Alexa 488; green) and LC3C (rabbit anti-LC3C; Alexa 594; red) were detected via dual staining. ( D ) HSp27 (mouse anti-HSp27; Alexa 488; green) and LC3C detection (rabbit anti-LC3C; Alexa 594; red) were also detected. Images were then captured using super resolution confocal laser scanning microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 10x and 63x magnification with oil immersion. Zoomed (2x) 3D versions of the 63x magnifications were obtained via the Imaris software. The range of z-stacks was kept consistent. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 200µm for 10x and 20 µm for 63x. Quantification of mean fluorescence intensity provided in Supplement. LC3C and HSp27 both were found to exhibit high levels of co-localization with each other and with P. g, as the Pearson coefficient was calculated to be .93 for LC3C and P. g and .85 for LC3C and HSp27 via the Imaris Software at the most severe state of disease.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: In Situ, Expressing, Microarray, Staining, Confocal Laser Scanning Microscopy, Software, Fluorescence

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Quantifications of Cross-Sectional Human Ex-Vivo Samples Support High Levels of P. gingivalis (P. g) , HSp27, and LC3C in Chronically Diseased Oral Tissues (i.e. Periodontitis). Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis patients were obtained using the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System so that the mean fluorescence intensity of ( A ) HSp27 ( B ) P. g and ( C ) LC3C could be calculated using ImageJ with JACoP Plugin. Data are presented as mean ± SD. Representative images from at least 5 different patients per group were used for quantitative analysis and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Ex Vivo, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 is a Critical Regulator in Pro-bacterial LC3C-Characterized Autophagy, Facilitating the Intracellular Autophagic Survival of P. gingivalis (P. g) and Influencing the Bacterial Symbiosis of the Oral Mucosa. The proposed diagram of the identified mechanisms of P. gingivalis persistence in GECs. ( A ) HSp27 is largely induced and spatially recruited by P. g invasion of the host cells. After the initial periods of cellular infection, the gradually growing secretion of the bacterial Nucleoside-diphosphate-kinase (Ndk) into the cytoplasmic space causes heightened activation of HSp27 (P-HSp27) via direct phosphorylation. P-HSp27 abrogates extracellular ATP (eATP)-induced antimicrobial Reactive-Oxygen-Species (ROS) production via increasing glutathione (GSH) levels. In parallel, P. g -mediated induction of HSp27 promotes the specific recruitment and lipidation of LC3C, an isomer of the LC3 autophagosomal structural molecule, which is strictly dependent upon the large presence and the strong antioxidant activity of HSp27. LC3C and HSp27 partner in a stepwise manner, 1) their coupling drives the formation of Beclin1/ATG14 induction, 2) where the temporally increased phosphorylation of HSp27 by P. g Ndk both strengthens the P-HSP27 and LC3C partnering and shifts the confirmation of the LC3C tail so that LC3C cannot be successfully further cleaved by ATG4. ( B ) P-HSp27 and LC3C become increasingly assembled to the ATG14-Incorperated Nucleation Complex, where they prolong the complex’s formation and result in the accumulation of the complex on forming autophagic membranes. Thus, the strengthened partnering between P-HSp27 and LC3C is the proposed mechanism for inhibiting the autolysosomal fusion of P. g- specific autophagosomes, which is also controlled by the host cell redox homeostasis ( C ) Autophagic P. g does not undergo lysosomal degradation and is instead able to survive, multiply and subsequently intercellularly spread to neighboring cells to propagate. ( D ) Thus, the non-canonical, pro-bacterial autophagic events create a favorable and protected cellular environment for P. g , thereby establishing long-term intracellular bacterial persistence. The chronic colonization of P. g in the epithelia can lead to host-microbial dysbiosis in oral mucosa and systemic disorders.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Infection, Activation Assay, Phospho-proteomics, Antioxidant Activity Assay

Journal: STAR Protocols

Article Title: Protocol for isolation and proteostatic analysis of sub-populations of spermatogenic cells in mouse

doi: 10.1016/j.xpro.2022.101398

Figure Lengend Snippet:

Article Snippet: Taq DNA polymerase , TIANGEN , Cat#ET101.

Techniques: Recombinant, Reverse Transcription, Saline, Protease Inhibitor, Purification, Bicinchoninic Acid Protein Assay, Software, Laser-Scanning Microscopy, Microscopy, Imaging, Membrane, Cell Culture

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet:

Article Snippet: Biotin-SP-affinipure goat anti-rabbit IgG, F(ab’)2 , Jackson Immuno Research Laboratories , Cat# 111-065-047; RRID: AB_2337964.

Techniques: Virus, Recombinant, Electron Microscopy, RNAscope, Multiplex Assay, Gene Expression, Plasmid Preparation, Sequencing, Positive Control, Negative Control, Software, RNA Sequencing, Transmission Assay, Microscopy, Laser-Scanning Microscopy, Imaging