Journal: Autophagy

Article Title: Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

doi: 10.1080/15548627.2024.2330043

Figure Lengend Snippet: S. pneumoniae infections induced lung injuries possibly through the disruption of tight junctions and AEB integrity. (A-F) Balb/c mice were either control-treated or intranasal-infected by D39 at a CFU of 5 × 10 8 per mouse, and the tissues and whole-cell extracts were collected at 24 h after intranasal infection with bacteria. (A) bacterial burdens in the lungs, brains, and blood were determined respectively ( n = 5). (B) Representative histological views of the lungs of mice by H&E staining. Arrows showed examples of disordered alveolar structures and neutrophil infiltration areas. Scale bars: 50 μm. (C and D) cytokine expressions of lung tissues were detected by cytokines array, and the fold changes compared to control were calculated according to gray levels. The indicated numbers from 1 to 10 in both C and D were corresponded to each other. (E and F) WB analysis of OCLN and LC3 levels of lung tissues at 24 hpi. (G) TEER changes of A549 cells in treating multiple dosages of D39 or PBS (mock) monitored by the electrical cell-substrate impedance sensing (ECIS) system. Data were collected and presented as mean ± SD from six replicated wells at each time point. (H) A549 cells were either control-treated or D39-infected at multiple moi (20, 50, and 100 moi), and whole-cell extracts were prepared at the respective time points (2, 4, and 8 hpi) for OCLN determination by WB. (I) Representative images of immunofluorescence staining of A549 cells infected with D39 for OCLN (red) co-stained with DAPI (blue). Scale bars: 20 μm.

Article Snippet: The A549 cells (ATCC, CCL-185), A549- ATG16L1 KO cells, A549-shCtrl cells, A549-sh BECN1 cells, A549-WT BECN1 cells, A549-BECN1 S93,96A cells, A549-HsLC3B-eGFP-MYC-TurboID cells, and A549-eGFP-MYC-TurboID cells (all of these cells were constructed in this study.

Techniques: Disruption, Control, Infection, Bacteria, Staining, Immunofluorescence

Journal: Autophagy

Article Title: Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

doi: 10.1080/15548627.2024.2330043

Figure Lengend Snippet: S. pneumoniae infections induced autophagy in A549 cells. (A and B) WB analysis of the LC3 and SQSTM1 protein expressions in A549 cells in response to the infection of 50 moi of D39 at the respective time points (1, 2, 3, 4, 5, 6, 8, and 10 hpi). (C) A549 cells were infected with D39 (moi of 50 at 4 hpi) in the presence or absence of CQ (50 μM, 3 h) before LC3 and SQSTM1 WB analysis. (D-F) A549 cells were infected by 50 moi of D39 for 4 h, followed by fixation and imaging preparation. (D) the visualization of the ultrastructure of A549 cells via TEM. In the images, membrane-like vesicles in D39-infected cells were observed. Arrows indicated membrane-like autophagosomes and autolysosomes. Scale bars: 2 μm, 500 nm. (E) immunofluorescence microscopy visualization of LC3 puncta in D39-infected A549 cells. Confocal images of ptfLC3-transfected A549 cells were infected with D39 or treated for 12 h with 100 nM rapa. Scale bars: 10 μm. (F) images of mCherry-LC3 fluorescent dots (red), FITC-LAMP1 fluorescence (green), and the overlay with DAPI (blue), which shows colocalization of mCherry- and FITC-positive puncta. The rapa-treated group was set as the positive control. Scale bars: 10 μm.

Article Snippet: The A549 cells (ATCC, CCL-185), A549- ATG16L1 KO cells, A549-shCtrl cells, A549-sh BECN1 cells, A549-WT BECN1 cells, A549-BECN1 S93,96A cells, A549-HsLC3B-eGFP-MYC-TurboID cells, and A549-eGFP-MYC-TurboID cells (all of these cells were constructed in this study.

Techniques: Infection, Imaging, Membrane, Immunofluorescence, Microscopy, Transfection, Fluorescence, Positive Control

Journal: Autophagy

Article Title: Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

doi: 10.1080/15548627.2024.2330043

Figure Lengend Snippet: S. pneumoniae infections induced autophagosomal degradation of TJ protein OCLN in A549 cells. (A) after the D39 infections, the total OCLN in the A549 cells was quantified by WB analyses. Blockage of autophagy by CQ caused an accumulation of OCLN in A549 cells. (B) after the knockdown of BECN1 in A549 cells, the autophagy activation and OCLN degradation were relieved under S. pneumoniae infections. (C) immunofluorescence microscopy visualization of the colocalization of Cy3-OCLN fluorescence (red) and the FITC-LAMP1 fluorescence (green) in D39-infected A549 cells. Scale bars: 10 μm. (D) schematic overview of autophagosome targeting and labeling using TurboID N-terminally fused to HsLC3B. A: autophagosome; N: nucleus. (E) A549 cells expressing HsLC3B-eGFP-MYC-TurboID were grown in the presence of DOX (12 h), BafA 1 (2 h), D39 (4 h), biotin (1 h) and ATP (1 h) followed by fixation and immunolabeling with biotin-546, the colocalization of HsLC3B-eGFP-MYC-TurboID chimeras with biotinylated molecules were observed. Scale bars, 5 μm. (F) homogenates from HsLC3B-eGFP-MYC-TurboID chimera expressing A549 cells grown in the presence of DOX (12 h), BafA 1 (2 h), D39 (4 h), biotin (1 h) and ATP (1 h) were left untreated or incubated with protK, Triton X-100, or both followed by immunoblotting. (G and H) lysates from the above protK-treated homogenates were immunoprecipitated with streptavidin magnetic beads, and the whole cell lysates (H: input) and beads-bound proteins from protK-protected proteins (G: IP) were respectively analyzed by immunoblotting. (I and J) A549 cells transiently expressing eGFP-SQSTM1, eGFP-LC3B, or eGFP-empty infected with D39 for 4 h were subjected to IP and assayed using an anti-eGFP antibody and protein A+G agarose. The total proteins (J: input) and bound proteins (I: IP) were analyzed by immunoblotting.

Article Snippet: The A549 cells (ATCC, CCL-185), A549- ATG16L1 KO cells, A549-shCtrl cells, A549-sh BECN1 cells, A549-WT BECN1 cells, A549-BECN1 S93,96A cells, A549-HsLC3B-eGFP-MYC-TurboID cells, and A549-eGFP-MYC-TurboID cells (all of these cells were constructed in this study.

Techniques: Knockdown, Activation Assay, Immunofluorescence, Microscopy, Fluorescence, Infection, Labeling, Expressing, Immunolabeling, Incubation, Western Blot, Immunoprecipitation, Magnetic Beads

Journal: Autophagy

Article Title: Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

doi: 10.1080/15548627.2024.2330043

Figure Lengend Snippet: pEvs were internalized into A549 cells to activate autophagy and disrupt the integrity of AEB. (A) detection of intracellular pKH67-labeled (green) pEvs in A549 cells with zeiss LSM800 confocal laser scanning microscope at the indicated time points after exposure to 100 μg/mL pEvs. Scale bars: 20 μm. (B) fluorescence intensity analysis of intracellular DiO-labeled pEvs (excitation: 484 nm, emission: 501 nm); DiO-treated PBS was used in the mock group. (C) TEER changes of A549 monolayer in the treatment of multiple dosages of pEvs or PBS (mock) monitored by the ECIS system. All the data was collected and presented as mean ± SD from three replicated wells at each time point. (D and F) WB analysis of the LC3 and OCLN protein expressions in A549 cells in response to multiple dosages of pEvs. (E and G) A549 cells were incubated with pEvs (100 μg/mL) in the presence or absence of CQ (50 μM, 3 h) before WB analysis of LC3 (E) and OCLN (G).

Article Snippet: The A549 cells (ATCC, CCL-185), A549- ATG16L1 KO cells, A549-shCtrl cells, A549-sh BECN1 cells, A549-WT BECN1 cells, A549-BECN1 S93,96A cells, A549-HsLC3B-eGFP-MYC-TurboID cells, and A549-eGFP-MYC-TurboID cells (all of these cells were constructed in this study.

Techniques: Labeling, Laser-Scanning Microscopy, Fluorescence, Incubation

Journal: Autophagy

Article Title: Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

doi: 10.1080/15548627.2024.2330043

Figure Lengend Snippet: pEvs-derived StkP activated autophagy by inducing BECN1 phosphorylation and resulted in autophagic degradation of epithelial OCLN in A549 cells. (A) purified pEvs from three independent biological replicates were analyzed by LC/MS. A total of 605 proteins were common to all three groups. The top 50 proteins ranked by average intensity (4D label-free) were listed. (B) lysates from A549 cells transiently expressing eGFP-tagged pEvs cargo proteins or eGFP-empty were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, eGFP, OCLN, or ACTB. (C) microscopy visualization of mCherry-LC3 fluorescent dots (red), LC3 puncta in eGFP-StkP- or eGFP-expressing A549 cells. Scale bar: 15 μm. (D) immunostaining of Cy3-OCLN (red) in A549 cells instantly expressing eGFP-StkP or eGFP alone. Scale bars: 20 μm. (E and F) lysates from A549 cells transiently expressing eGFP-StkP or eGFP and HA-BECN1 were performed IP with an anti-eGFP antibody and protein A+G agarose (E, upper two panels) or only an anti-HA magnetic beads (E, lower two panels). The total proteins (F: input) and respective bound proteins were analyzed by immunoblotting (E: IP). (G) WB detection of BECN1 phosphorylation (Ser93 and Ser96) level in A549 cells overexpressing eGFP or eGFP-stkP. (H) after overexpressing StkP in A549 cells, LC3 and OCLN were quantified by WB analyses. Blockage of autophagy by CQ caused increased expression of both LC3-II and OCLN. (I) WT BECN1 or BECN1 S93,96A were overexpressed in BECN1 knockdown A549 cells (by shRNA), and western blotting analysis of OCLN expression was performed in WT BECN1 or BECN1 S93,96A mutated A549 cells following introduction with eGFP or eGFP-StkP.

Article Snippet: The A549 cells (ATCC, CCL-185), A549- ATG16L1 KO cells, A549-shCtrl cells, A549-sh BECN1 cells, A549-WT BECN1 cells, A549-BECN1 S93,96A cells, A549-HsLC3B-eGFP-MYC-TurboID cells, and A549-eGFP-MYC-TurboID cells (all of these cells were constructed in this study.

Techniques: Derivative Assay, Phospho-proteomics, Purification, Liquid Chromatography with Mass Spectroscopy, Expressing, SDS Page, Western Blot, Microscopy, Immunostaining, Magnetic Beads, Knockdown, shRNA

Journal: Autophagy

Article Title: Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

doi: 10.1080/15548627.2024.2330043

Figure Lengend Snippet: StkP-laden pEvs contributed to S. pneumoniae virulence and bacterial dissemination by disrupting AEB integrity. (A) Balb/c mice were intranasally challenged with D39-WT, D39-Δ stkP , and D39-C-Δ stkP at 5 × 10 8 CFU in 40 μL PBS; bacterial infected mice were continuously observed to obtain survival data ( n = 10). (B) bacterial burdens in the lung tissues and blood were determined at 24 hpi ( n = 5). (C) Representative histological views of the lung tissues of mice at 24 hpi by H&E staining. Scale bars: 100 μm. (D) WB analysis of the lung lysates at 24 hpi for indicated antibodies: BECN1, p-BECN1-Ser93,Ser96, LC3, and OCLN. (E) the expression and localization of LC3 and OCLN were observed in the mice’s lung tissues by immunofluorescent staining. Scale bars: 20 μm. Arrows indicated colocalization of increased LC3 and OCLN in lung tissues. (F) TEER changes of A549 monolayer incubated by 100 μg/mL of pEvs derived from D39-WT, D39-Δ stkP , and D39-C-Δ stkP respectively monitored by the ECIS system. All the data was collected and presented as mean ± SD from six replicated wells at each time point. (G) WB analysis of indicated protein in A549 cells treated by pEvs from D39-WT, D39-Δ stkP , and D39-C-Δ stkP , respectively. (H) OCLN integrity observation in different pEvs-treated A549 cells by confocal laser microscopy. Scale bars: 20 μm. (I) confocal imaging of LC3 puncta after incubation with 100 μg/mL of pEvs derived from D39-WT, D39-Δs tkP , or D39-C-Δs tkP , respectively. Scale bars: 10 μm.

Article Snippet: The A549 cells (ATCC, CCL-185), A549- ATG16L1 KO cells, A549-shCtrl cells, A549-sh BECN1 cells, A549-WT BECN1 cells, A549-BECN1 S93,96A cells, A549-HsLC3B-eGFP-MYC-TurboID cells, and A549-eGFP-MYC-TurboID cells (all of these cells were constructed in this study.

Techniques: Infection, Staining, Expressing, Incubation, Derivative Assay, Microscopy, Imaging

Journal: bioRxiv

Article Title: Key role of vimentin in the organization of the primary cilium

doi: 10.1101/2024.01.17.576004

Figure Lengend Snippet: (A) Scheme of the architecture and main elements of the primary cilium. In quiescent cells the centrosome evolves to give rise to the primary cilium. The mother centriole forms the basal body from which nine microtubule triplets extend forming the internal cilium scaffold or axoneme. At distal sections the microtubule triplets become doublets. Tubulin is heavily acetylated along the length of the cilium. The two centrioles are surrounded by the pericentriolar material (PCM), which contains elements important for ciliogenesis, including Rab GTPases in charge of the traffic of building elements for the cilium, γ-tubulin and pericentrin (please see text for details). (B) A549 cells were cultured during 7 days after passage and vimentin (antibody 84.1) and acetylated tubulin (ac-tubulin) were detected by immunofluorescence. The upper row presents overall projections of the individual and merged channels. The area of interest is enlarged in the medium row, which depicts single confocal sections, where several structures compatible with cilia are marked by arrows. One of the cilia is shown in the lower row in more detail. Scale bars, 20 µm. (C) A stack of the single sections obtained every 0.5 µm is shown to illustrate the upward projection of the cilium. Images are rotated 90° counter-clock wise with respect to (B). (D) A549 cells were cultured and stained with the antibodies 84.1 and anti-acetylated tubulin, as above. Images were obtained with the Lightning module of the Leica SP8 microscope. Single channels and merged images are shown. The region of interest denoted by the dotted square is enlarged at the right (scale bars, 10 and 5 μm, respectively). (E) Images of a cilium were obtained by STED superresolution microscopy and a 3D reconstruction of this structure is provided in the right image to illustrate the close intertwining of vimentin and acetylated tubulin (scale bars, 5 and 2 μm, respectively). (F) The cilia fraction was isolated and analyzed by electron microscopy; scale bar, 500 nm. (G) Total lysates from A549 cells and the cilia fraction were analyzed by SDS-PAGE and western blot with the indicated antibodies. (H) The cilia fraction was resuspended in hypotonic buffer and soluble (S100) and insoluble (P100) fractions, analyzed by SDS-PAGE and western blot with anti-vimentin. (I) The cilia fraction was subjected to proteomic analysis and the parameters corresponding to the identification of vimentin are shown.

Article Snippet: A549 VIM KO cells were obtained by disruption of the VIM gene with the CRISPR double nickase technology using the Vimentin Double Nickase Plasmid (h) from Santa Cruz Biotechnology (sc-400035-NIC), following the instructions of the manufacturer.

Techniques: Cell Culture, Immunofluorescence, Staining, Microscopy, Isolation, Electron Microscopy, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Key role of vimentin in the organization of the primary cilium

doi: 10.1101/2024.01.17.576004

Figure Lengend Snippet: (A) A549 cells cultured as above were fixed and processed for immunofluorescence. Total vimentin was detected with the SP20 antibody and phospho-specific antibodies were used for detection of pSer39, pSer56 or pSer72-vimentin. In the lower row, signals compatible with primary cilia are marked by arrows. (B) Localization of pSer56-vimentin at primary cilia was confirmed by staining of acetylated tubulin. Images shown are single confocal sections. Several structures positive for both proteins are marked by arrows. Bars, 20 μm.

Article Snippet: A549 VIM KO cells were obtained by disruption of the VIM gene with the CRISPR double nickase technology using the Vimentin Double Nickase Plasmid (h) from Santa Cruz Biotechnology (sc-400035-NIC), following the instructions of the manufacturer.

Techniques: Cell Culture, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Key role of vimentin in the organization of the primary cilium

doi: 10.1101/2024.01.17.576004

Figure Lengend Snippet: (A) A549 cells were cultured for an initial 5-day period and then subjected to a starvation condition for two days. pSer56-vimentin and acetylated tubulin were detected by immunofluorescence and STED microscopy. Areas of interest denoted by dotted boxes are enlarged at the right (scale bars, upper image 10 µm, lower image, 5 µm). (B) Immunostaining of Rab11 and acetylated tubulin to show the presence of Rab11 at the base of the primary cilium. Inset is enlarged at the bottom images. Images shown are single sections. (C) The presence of the pSer56-vimentin signal at the pericentriolar material is assessed by immunofluorescence using Rab11 as a marker of this structure. The area of interest in the merged image is enlarged at right and at the bottom. Further detail of one of the cilia is depicted in insets. Images shown are single sections. (D) Detection of total vimentin in relation to the pericentriolar material. Images at the right depict composite images of one cilium stained with anti-Rab11 (green), anti-acetylated tubulin (magenta), and for total vimentin (84.1 antibody, grayscale). Nuclei were counterstained with DAPI. Images at the right show enlarged views of the area of interest as single channels for the various signals and a merged image (bottom right). Note the presence of vimentin at the cilium and the absence of enrichment of its signal at the pericentriolar material region highlighted by the Rab11 signal. Images shown are single sections. Scale bars, left images, 10 µm, right images, 5 µm.

Article Snippet: A549 VIM KO cells were obtained by disruption of the VIM gene with the CRISPR double nickase technology using the Vimentin Double Nickase Plasmid (h) from Santa Cruz Biotechnology (sc-400035-NIC), following the instructions of the manufacturer.

Techniques: Cell Culture, Immunofluorescence, Microscopy, Immunostaining, Marker, Staining

Journal: bioRxiv

Article Title: Key role of vimentin in the organization of the primary cilium

doi: 10.1101/2024.01.17.576004

Figure Lengend Snippet: MEF wt or Vim(-/-) (A) or A549 wt and VIM KO cells (B) were stained for Rab11 and acetylated tubulin, as indicated. Single channels or merged images or overall projections are shown. The areas of interest marked by dotted boxes are enlarged in the right panels. The proportion of cells displaying a defined pericentriolar material (PCM) region, as highlighted by Rab11 staining are shown on the graphs at far right. Results shown are average values ± SEM of the proportions obtained in the analysis of ten (A) and six (B) fields, with approximately 50 cells each. ****p<0.0001 by unpaired t -test.

Article Snippet: A549 VIM KO cells were obtained by disruption of the VIM gene with the CRISPR double nickase technology using the Vimentin Double Nickase Plasmid (h) from Santa Cruz Biotechnology (sc-400035-NIC), following the instructions of the manufacturer.

Techniques: Staining

Journal: Molecular Oncology

Article Title: SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis

doi: 10.1002/1878-0261.12959

Figure Lengend Snippet: si‐hSMAC‐A treatment attenuates A549 lung cancer tumour xenograft growth and altered phospholipid levels. (A) Tumour growth of the A549 cell xenograft in immunodeficient 6‐week‐old male athymic nude mice. Mice were treated twice a week intratumourally with si‐hSMAC‐A (350 and 700 n m ) or with nontargeted si‐RNA (si‐NT, 350 n m ) ( n = 8 mice/group). Treatment was initiated when the average tumour volume in each group reached ˜ 50 mm 3 . Results are presented as the mean tumour volume ± SEM. (B,C) Immunoblot (B) and quantitative analysis (C) of SMAC, VDAC1 and citrate synthase (CS) in si‐hSMAC‐TTs and si‐NT‐TTs samples ( n = 6 mice/group). (D‐F) IHC stained of si‐hSMAC‐ or si‐NT‐TTs sections for SMAC (D), Ki‐67 (E), and their quantitative analysis (F) ( n = 6 mice/group). Scale bars represent 50, 25 or 15 µm as indicated. (G) PL, PC and PE analysis was carried out as described in section ( n = 6 mice per group). Results are the means ± SEM. P values were calculated using two‐sided Student's t ‐test, ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: SMAC‐expressing and SMAC‐KO A549 cells represent comparative models to investigate SMAC function in regulating phospholipid synthesis.

Techniques: Western Blot, Staining

Journal: Molecular Oncology

Article Title: SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis

doi: 10.1002/1878-0261.12959

Figure Lengend Snippet: SMAC knockout in A549 cells by Crisper/Cas inhibits cell proliferation that could be restored upon SMAC re‐expression. (A) Schematic presentation of CRISPR/Cas9 mediates knockout. (B‐D) Representative IF images (scale bars = 20 µm) (B) their quantification (C) of SMAC in control and CRISPR/Cas9‐generated SMAC‐deficient A549 cells and HEK‐293T cells, and immunoblotting (D) stained with anti‐SMAC antibodies. (E) Cell proliferation in SMAC knockout A549 and HEK‐293T cells ( n ‐4) as analysed using the SRB method ( n = 4). (F,G) A549 cells expressing SMAC were transfected with control plasmid, and SMAC knockout A549 cells were transfected with plasmid pCDNA3.1 (0.5 or 1 µg DNA) encoding full‐length SMAC. After 48 h cells were analysed by immunoblotting for SMAC expression (F) and cell proliferation ( n = 4) (G). Results are the means ± SEM, P values were calculated using two‐sided Student's t ‐test, ** P ≤ 0.01; *** P ≤ 0.001; NS, nonsignificant.

Article Snippet: SMAC‐expressing and SMAC‐KO A549 cells represent comparative models to investigate SMAC function in regulating phospholipid synthesis.

Techniques: Knock-Out, Expressing, CRISPR, Generated, Western Blot, Staining, Transfection, Plasmid Preparation

Journal: Molecular Oncology

Article Title: SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis

doi: 10.1002/1878-0261.12959

Figure Lengend Snippet: Subcellular morphological alterations including ER‐mitochondria contact sites, as induced by reduction of SMAC levels. (A,B) Representative transmission electron micrographs of sections from si‐NT‐TTs (A) and si‐hSMAC‐A‐TTs (B) from A549 xenografts carried out as described previously . Various membrane organelles as intracellular vesicles containing surfactant‐accumulating lamellar bodies MAM‐like structures (ER‐associated mitochondria) are seen in the si‐NT‐TTs (Aa‐c), but not in si‐hSMAC‐A‐TTs, showing enrichment in the mitochondria (m), ER and nucleus (Nu). White (b) and red (e,f) arrows point to lamellar bodies and ER, respectively. Mitochondria number in si‐NT‐TTs and si‐hSMAC‐A‐TTs per EM section was 1‐3 and 8‐15, respectively. Scale bars represent 2 or 0.5 µm as indicated. (C,D) IF staining for IP 3 R (red) and VDAC1 (green) in si‐NT‐TTs and si‐hSMAC‐A‐TTs sections. Images were captured by confocal microscope ( n = 5, scale bar represents 10 µm). (C) and subjected to quantitative analysis ( n = 5) (D). (E,F) si‐NT‐TTs and si‐hSMAC‐TTs were subjected to in‐situ PLA to test for close association between VDAC1 (OMM) and IP 3 R (ER) (MAM) using specific antibodies. The ligation products appear in red; nuclei were DAPI‐stained. Images at high magnification (Ea,b) (scale bars represent 20, 5 µm as indicated) and ligation product quantification (F) are also shown. Results are the means ± SEM, P values were calculated using two‐sided Student's t ‐test, *** P ≤ 0.001.

Article Snippet: SMAC‐expressing and SMAC‐KO A549 cells represent comparative models to investigate SMAC function in regulating phospholipid synthesis.

Techniques: Transmission Assay, Staining, Microscopy, In Situ, Ligation

Journal: Molecular Oncology

Article Title: SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis

doi: 10.1002/1878-0261.12959

Figure Lengend Snippet: SMAC knockout in A549, but not in HEK‐293T cells reduces PL and PC, and increases PE. (A) Schematic presentation of PE reaction with DSB‐3 and obtained fluorescence signals used for PSD activity analysis. (B) PLs were extracted from the indicated cells and analysed for total PL, PC and PE levels as described in the section. (C) Schematic presentation of PS imported from the ER into the mitochondria at the MAM and its conversion to PE by PSD. (D,E) PSD activity (D) and PE levels (E) in control and SMAC‐KO, A549 and HEK‐293T cells analysed as described in the section. (F,G) Control and SMAC‐knockout A549 cells and their mitochondria‐free and mitochondria‐enriched subfractions (obtained as described in the section) were subjected to immunoblotting (F) or PSD activity assay (G). Total PL, PC and PE as analysed in SMAC‐KO A549 cell extract, mitochondria‐free and mitochondria‐enriched fractions relative to their levels in SMAC‐expressing A549 cells (H). Results are the mean ± SEM ( n = 3); P values were calculated using two‐sided Student's t ‐test, * P ≤ 0.05, ** P ≤ 0.01; *** P ≤ 0.001, NS, nonsignificant.

Article Snippet: SMAC‐expressing and SMAC‐KO A549 cells represent comparative models to investigate SMAC function in regulating phospholipid synthesis.

Techniques: Knock-Out, Fluorescence, Activity Assay, Western Blot, Expressing

Journal: Molecular Oncology

Article Title: SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis

doi: 10.1002/1878-0261.12959

Figure Lengend Snippet: SMAC is colocalized with PSD, interacts with and negatively regulates PSD activity. (A) Representative IF images for SMAC and PSD colocalization in A549 cells. Scale bars represent 10, 2.5 µm, as indicated. (B,C) PLA assay showing direct interaction between SMAC and PSD in A549 cells (scale bar represent 10 µm). (B) and quantitative analysis ( n = 3) of the ligation product (C) Results are the mean ± SEM ( n = 3) P values were calculated using two‐sided Student's t ‐test, ** P ≤ 0.01. (D‐G) Immunoblot (D) and quantitative analysis cells (E) of PSD and SMAC expression levels relative to WI‐38 ( n = 3), PSD activity ( n = 6) (F) and PE levels ( n = 6) (G) were analysed in the indicated cell lines. (H) Purified PSD and SMAC used in this study. (I) PSD interaction with SMAC analysed using the MST method ( n = 4). Purified SMAC was fluorescently labelled with the NanoTemper BLUE protein‐labelling kit. SMAC (1 µ m ) was incubated with purified PISD (78–625 n m ) for 30 min at 37 °C, then thermophoresis was measured as described in the section. K d = 246 ± 29 n m ( n = 3). (J) Inhibition of PSD activity by SMAC, as measured using the DSB‐3 method ( n = 4). Results are the mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; NS, nonsignificant.

Article Snippet: SMAC‐expressing and SMAC‐KO A549 cells represent comparative models to investigate SMAC function in regulating phospholipid synthesis.

Techniques: Activity Assay, Ligation, Western Blot, Expressing, Purification, Incubation, Inhibition

Journal: Molecular Oncology

Article Title: SMAC/Diablo controls proliferation of cancer cells by regulating phosphatidylethanolamine synthesis

doi: 10.1002/1878-0261.12959

Figure Lengend Snippet: PSD‐interacting peptides bind to PSD, inhibiting its activity, and when cell‐penetrating and mitochondria‐ or nucleus‐targeted, inhibit cell proliferation. (A) Fluorescently‐labelled purified PSD (0.25 µ m ) was incubated with 2F3 (●) or 1C11 (○) peptide (1–10 µ m ) for 30 min at 37 °C; then, MST was used and revealed K d of 3.0 µ m for both peptides ( n = 3). (B) Inhibition of PSD activity by the 2F3 (●) or 1C11 (○) peptide. PSD activity was measured using the DSB‐3 method. (C‐E) Cell proliferation inhibition following incubation of A549 cells with the indicated mitochondria‐ or nucleus‐targeted peptides for 24 h in a serum‐free medium, and cell proliferation was assayed using the SRB method. Results are the means ± SEM ( n = 3). (F) A549 cells were incubated for 90 min with 5 µ m of the mitochondria‐ or nucleus‐targeted FITC‐labelled peptides, immunostained with anti‐SMAC antibodies and with DAPI and visualized by confocal microscope for subcellular localization. (a) and (b) present higher magnification, white arrows point to possible nuclear localization of the peptide, and blue arrows to its colocalization with mitochondria marker ( n = 3). Scale bars represent 20, 10 µm as indicated.

Article Snippet: SMAC‐expressing and SMAC‐KO A549 cells represent comparative models to investigate SMAC function in regulating phospholipid synthesis.

Techniques: Activity Assay, Purification, Incubation, Inhibition, Microscopy, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PGAM5 is a key driver of mitochondrial dysfunction in experimental lung fibrosis

doi: 10.1007/s00018-019-03133-1

Figure Lengend Snippet: PGAM5 drives bleomycin-induced cytotoxicity and mitochondrial dysfunction in human pulmonary epithelial cells. A549 (a–d) and A549 PGAM5-KO (a–e) cells were treated either with bleomycin 100 µg/ml (a, b, d, e) or bleomycin 200 µg/ml (d) and analyzed over the course of 48 h (a), respectively, after an interval of 48 h (b, d, e). Depictions are representative of three independent experiments with similar results. Untreated cells of each modality served as controls. (a) X-celligence. Normalized cell index (NCI) normalized to the time of bleomycin addition. Each curve represents multiple measurements (n = 4 for untreated, n = 6 for treated modalities). Two-way ANOVA with Tukey’s test (***p < 0.001 comparing bleomycin-treated A549 PGAM5-KO to bleomycin-treated A549). Displayed are mean and error ± SD. b Light microscopy. Left panel: A549 and A549 PGAM5-KO cells (bar 500 µm). Right panel: A549 PGAM5-KO cells either transfected with a transfection control or with a PGAM5 plasmid to induce PGAM5 expression (bar 100 µm). c PGAM5 deficiency (upper panel) and efficient vector-based PGAM5-expression in genetically deficient A549 PGAM5-KO cells (lower panel) was confirmed using immunoblotting. β-Actin served as loading control. d, e JC-1 experiments: healthy cells (intact ΔΨ) emit a red signal (JC-1 aggregates). Cells with depolarized ΔΨ emit a green signal (JC-1 monomers). An increased green-to-red-ratio indicates a higher percentage of depolarized cells. Graphs are Tukey box plots. d Representative JC-1 IF microscopy (bleomycin 100 µg/ml, bar 75 µm). Cumulative quantification: > 900 events per modality were assessed. Two-way ANOVA with Tukey’s test (***p < 0.001). e A549 PGAM5-KO cells were analyzed in comparison to A549 PGAM5-KO cells with vector-based PGAM5 expression (via transfected plasmid analogous to Fig. 2b and c) employing JC-1 IF microscopy. Representative JC-1 IF microscopy (upper panel: untreated, bar 50 µm; lower panel: bleomycin 100 µg/ml, bar 75 µm). Quantification: > 1500 events of each modality were assessed. Two-way ANOVA with Tukey’s test (***p < 0.001)

Article Snippet: Immunoblotting Proteins were extracted either from whole cell lysate or mitochondrial fraction isolate of A549 and A549 PGAM5-KO cells using Cell Lysis Buffer (9803, Cell Signaling Technologies) with added Proteinase-Inhibitors (complete tablets mini, 04 693 159 001, Roche) and Phosphatase-Inhibitors (Phospho Stop, 04 906 837 001, Roche).

Techniques: Light Microscopy, Transfection, Control, Plasmid Preparation, Expressing, Western Blot, Microscopy, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PGAM5 is a key driver of mitochondrial dysfunction in experimental lung fibrosis

doi: 10.1007/s00018-019-03133-1

Figure Lengend Snippet: PGAM5 mediates structural damage of mitochondria in human pulmonary epithelial cells after bleomycin treatment. A549 and A549 PGAM5-KO cells were treated with bleomycin 100 µg/ml (a, c, d) or bleomycin 200 µg/ml (b, c, d) for 48 h unless stated otherwise. Depictions are representative of three independent experiments (a, c), one iteration per time point (b, 24 h results not shown) or one measurement series (d) with similar results. Nuclei were counterstained with Hoechst 33342. (a–c) Confocal immunofluorescence images (a) PGAM5. Right column: overlay of fluorescence images with cell contour outlines based on bright-field overlay (bar 20 µm; bright field not shown). (b) PGAM5 and TOMM20 co-localization (bar 10 µm). (c) TOMM20 visualization. Representative images of TOMM20 staining (bleomycin 100 µg/ml; 24 h, 48 h; bar 20 µm). Quantification (after 48 h): > 140 cells per modality were assessed. Tukey box plot. Two-way ANOVA with Tukey’s test (*p < 0.05/***p < 0.001). d Independent TEM images. Overview (upper panel): arrowheads mark different mitochondrial subtypes: elongated/branched (green) and swollen (red). Details (lower panel): red arrows mark mitochondrial membrane alterations

Article Snippet: Immunoblotting Proteins were extracted either from whole cell lysate or mitochondrial fraction isolate of A549 and A549 PGAM5-KO cells using Cell Lysis Buffer (9803, Cell Signaling Technologies) with added Proteinase-Inhibitors (complete tablets mini, 04 693 159 001, Roche) and Phosphatase-Inhibitors (Phospho Stop, 04 906 837 001, Roche).

Techniques: Immunofluorescence, Fluorescence, Staining, Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: PGAM5 is a key driver of mitochondrial dysfunction in experimental lung fibrosis

doi: 10.1007/s00018-019-03133-1

Figure Lengend Snippet: PGAM5 disrupts mitochondrial function by mediating downstream mitophagy independently of mtROS levels. A549 and A549 PGAM5-KO cells (a–d) were treated with bleomycin 100 µg/ml (a–d) or bleomycin 200 µg/ml (a–c) and analyzed after 24 h. All results are representative of three independent experiments with similar results. Nuclei were counterstained with Hoechst 33342. a Mitochondrial ROS (mtROS) were measured by mitoSOX flow cytometry. Histogram: x-axis displays fluorescence signal intensity; y-axis displays events as percentage of the maximum. Graph: aligned dot plot with mean ± SD of relative mean fluorescence intensity (MFI). Two-way ANOVA (ns p ≥ 0.05). Statistical analysis is cumulative. b Western blot from whole cell lysate with immunoblotting of LC3B. β-Actin served as loading control. Bar chart with mean and SEM displays the pooled densitometry results of the LC3BII–LC3BI ratio (lower and upper band, respectively) after normalization to β-actin [for each genotype (A549/A549 PGAM5-KO): n = 2 “untreated”; n = 3 “BLM 100 µg/ml”; n = 2 “BLM 200 µg/ml”]. Two-way ANOVA (ns p ≥ 0.05). Statistical analysis is cumulative. c Western blot from isolated mitochondria with immunoblotting of LC3B. VDAC served as loading control. Bar chart with mean and SEM displays the pooled densitometry results of the LC3BII–LC3BI ratio (lower and upper band, respectively) after normalization to VDAC [for each genotype (A549/A549 PGAM5-KO): n = 2 “untreated”; n = 3 “BLM 100 µg/ml”; n = 3 “BLM 200 µg/ml”]. Two-way ANOVA with Tukey’s test (ns p ≥ 0.05/**p < 0.01). Statistical analysis is cumulative. d Confocal immunofluorescence images of PINK1 (bar 20 µm). e Immunofluorescence images of LC3B on murine lung sections of mice sacrificed 7 days after bleomycin challenge (same cohort as Fig. 1c). Scale bar 50 µm (upper panel, microscopy), scale bar 7.5 µm (lower panel, confocal microscopy)

Article Snippet: Immunoblotting Proteins were extracted either from whole cell lysate or mitochondrial fraction isolate of A549 and A549 PGAM5-KO cells using Cell Lysis Buffer (9803, Cell Signaling Technologies) with added Proteinase-Inhibitors (complete tablets mini, 04 693 159 001, Roche) and Phosphatase-Inhibitors (Phospho Stop, 04 906 837 001, Roche).

Techniques: Flow Cytometry, Fluorescence, Western Blot, Control, Isolation, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: Cell reports

Article Title: Critical role of Syk-dependent STAT1 activation in innate antiviral immunity.

doi: 10.1016/j.celrep.2020.108627

Figure Lengend Snippet: Figure 1. STAT1 is activated in a cytokine-in- dependent manner at the initial stage of some virus infections (A–C) A549 cells (A) or BMDM cells from wild-type (WT) mice (C) were infected by WSN for indicated times. These cell-culture supernatants (SNs) were used to stimulate the native cells for 45 min. Cell lysates were analyzed by western blotting (WB) with indicated antibodies (A and C), and viral NS1 mRNA levels were measured by RT-PCR (A). (B) Phos- phorylated STAT1 at Y701 (pSTAT1(Y701)) in (A) was quantitated by densitometry and normalized to STAT1 and control b-actin levels. In each experi- ment, the highest level of pSTAT1 is set to 100. Data represent means ± SD from three independent ex- periments. **p < 0.01. p.i., post-infection. NP is short for nucleoprotein. (D–F) Indicated cell lines were infected with SeV (D), ZIKV (E), and PRV (F) and stimulated by corre- sponding SNs, as described in (A), and analyzed by WB and RT-PCR to detect the levels of the indicated genes and proteins. PRV-gD is short for PRV glycoprotein D. (G) Immunoblot of WSN-infected A549 cells for the indicated times. (H) IFNAR1 KO-A549 or control (WT-A549) cells were transfected with siRNA for 48 h and then in- fected with WSN for 3 h or stimulated by IFN-b (0.1 mg/mL) for 45 min. pSTAT1 was analyzed by WB. si-NC is short for si-negtive control. (I) ELISA analysis of IFN-b levels in the SN from WSN-infected NIH/3T3 cells for the indicated times. Data shown represent means ± SD from three in- dependent experiments. **p < 0.01. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trizol Invitrogen Cat#: 15596018 plasmids expressing myristoylated kinases Addgene, originally fromWilliam Hahn and Jean Zhao Cat#: 1000000012 Critical commercial assays mouse IFN-a ELISA kit eBioscience Cat#: BMS6027 Mouse IFN-b ELISA kit BioLegend Cat#: 439407 Experimental models: cell lines 293T ATCC Cat#: CRL-11268 A549 ATCC Cat#: CCL-185 PK-15 ATCC Cat#: CCL-33 NIH/3T3 ATCC Cat#: CRL-1658 RIG-I KO A549 This study N/A Syk KO A549 This study N/A IFNAR1 KO A549 This study N/A MDCK ATCC Cat#: CRL-2935 BMDM This study N/A AEC This study N/A Experimental models: organisms/strains Mouse: RIG-I / From Prof. Zhugang Wang, Shanghai Jiao Tong University N/A Mouse: IFNLR1+/ Cyagen Co., Ltd, Guangzhou Cat#: KOCMP-242700-Ifnlr1-B6N Mouse: IFNAR1 / From Prof. Dan Portnoy, University of California N/A Mouse: IFNAR1 / IFNLR1 / This study N/A Mouse: STAT1+/ From Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences N/A Mouse: STAT1Y701F This study N/A Mouse: Sykflox/flox Jackson Laboratory Cat#: 017309 Mouse: Ubc-CreERT2 Shanghai Research Center of Southern Model Organisms N/A Mouse: Sftpc-IRES-iCre (iCre) GemPharmatech Co., Ltd N/A Oligonucleotides shRNA, sgRNA, and siRNA oligonucleotides are in Table S1 N/A N/A Recombinant DNA pSpCas9(BB)-2A-GFP (PX458) From Constance Ciaudo Addgene Plasmid #48138 Syk-PX458 This study N/A RIG-I-PX458 This study N/A IFNAR1-PX458 This study N/A pNL-Myc-tagged MAVS This study N/A pNL-Flag-tagged SykWT This study N/A pNL-Flag-tagged SykSH2 This study N/A pNL-Flag-tagged Sykkinase This study N/A Software and algorithms ImageJ NIH https://imagej.net/ImageJ DNAMAN Lynnon Biosoft N/A Cell Reports 34, 108627, January 19, 2021 e2

Techniques: Virus, Infection, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Critical role of Syk-dependent STAT1 activation in innate antiviral immunity.

doi: 10.1016/j.celrep.2020.108627

Figure Lengend Snippet: Figure 3. RIG-I/MAVS pathway regulates IAV-induced initial activation of STAT1 (A–E) In (A), mock-, INCB-, and CP-pretreated A549 cells were transfected with cellular RNA (C-RNA), virus-infected cellular RNA (VC-RNA), or viral genomic RNA (VG-RNA) for 90 min. pSTAT1 was analyzed by WB. (B, C, and E) MDA5-ablated A549 cells (B), RIG-I-ablated A549 cells (C), and BMDM cells from indicated mice (E) were infected with WSN or stimulated by corresponding SNs, as described in Figure 1A. pSTAT1 was analyzed by WB. (D) WB of indicated proteins in lungs of WT and RIG-I KO mice infected with WSN at the indicated times. (F) RT-PCR (upper panel) and WB (lower panel) were performed to detect the levels of indicated genes and proteins in WSN-infected, MAVS-knockdown cells. Plotted are the average levels from three independent experiments. (G) Whole-cell lysates (WCLs) of WSN-infected A549 cells were immunoprecipitated with anti-MAVS antibody, and the precipitated proteins were examined by immunoblotting. (H) BAY 11-7082-pretreated A549 cells (for 20 min) were infected with WSN for 3 h. pSTAT1 was analyzed by WB. Shown are representative data from three independent experiments. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trizol Invitrogen Cat#: 15596018 plasmids expressing myristoylated kinases Addgene, originally fromWilliam Hahn and Jean Zhao Cat#: 1000000012 Critical commercial assays mouse IFN-a ELISA kit eBioscience Cat#: BMS6027 Mouse IFN-b ELISA kit BioLegend Cat#: 439407 Experimental models: cell lines 293T ATCC Cat#: CRL-11268 A549 ATCC Cat#: CCL-185 PK-15 ATCC Cat#: CCL-33 NIH/3T3 ATCC Cat#: CRL-1658 RIG-I KO A549 This study N/A Syk KO A549 This study N/A IFNAR1 KO A549 This study N/A MDCK ATCC Cat#: CRL-2935 BMDM This study N/A AEC This study N/A Experimental models: organisms/strains Mouse: RIG-I / From Prof. Zhugang Wang, Shanghai Jiao Tong University N/A Mouse: IFNLR1+/ Cyagen Co., Ltd, Guangzhou Cat#: KOCMP-242700-Ifnlr1-B6N Mouse: IFNAR1 / From Prof. Dan Portnoy, University of California N/A Mouse: IFNAR1 / IFNLR1 / This study N/A Mouse: STAT1+/ From Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences N/A Mouse: STAT1Y701F This study N/A Mouse: Sykflox/flox Jackson Laboratory Cat#: 017309 Mouse: Ubc-CreERT2 Shanghai Research Center of Southern Model Organisms N/A Mouse: Sftpc-IRES-iCre (iCre) GemPharmatech Co., Ltd N/A Oligonucleotides shRNA, sgRNA, and siRNA oligonucleotides are in Table S1 N/A N/A Recombinant DNA pSpCas9(BB)-2A-GFP (PX458) From Constance Ciaudo Addgene Plasmid #48138 Syk-PX458 This study N/A RIG-I-PX458 This study N/A IFNAR1-PX458 This study N/A pNL-Myc-tagged MAVS This study N/A pNL-Flag-tagged SykWT This study N/A pNL-Flag-tagged SykSH2 This study N/A pNL-Flag-tagged Sykkinase This study N/A Software and algorithms ImageJ NIH https://imagej.net/ImageJ DNAMAN Lynnon Biosoft N/A Cell Reports 34, 108627, January 19, 2021 e2

Techniques: Activation Assay, Transfection, Virus, Infection, Reverse Transcription Polymerase Chain Reaction, Knockdown, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: Critical role of Syk-dependent STAT1 activation in innate antiviral immunity.

doi: 10.1016/j.celrep.2020.108627

Figure Lengend Snippet: Figure 4. Syk is identified as a key kinase mediating initial activation of STAT1 induced by IAV infection (A and B) The pSTAT1 levels in WSN-infected Yes (A) or Syk (B) knockdown A549 cells were analyzed at indicated time by WB. (C and D) Shown is an immunoblot of WSN-infected (C), CA04-infected (D, left) or PR8-infected (D, right) Syk knockout (Syk KO-A549) or control cells, probed with indicated antibodies. Plotted are the average levels from three independent experiments. (E) WSN-infected indicated cells, stably expressing empty vector (EV) or FLAG-Syk, were probed by WB with indicated antibodies. (F) The pSTAT1 levels in indicated cells stimulated by IFN-b (0.1 mg/mL) (left) or IL-29 (0.1 mg/mL) (right) were analyzed by WB.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trizol Invitrogen Cat#: 15596018 plasmids expressing myristoylated kinases Addgene, originally fromWilliam Hahn and Jean Zhao Cat#: 1000000012 Critical commercial assays mouse IFN-a ELISA kit eBioscience Cat#: BMS6027 Mouse IFN-b ELISA kit BioLegend Cat#: 439407 Experimental models: cell lines 293T ATCC Cat#: CRL-11268 A549 ATCC Cat#: CCL-185 PK-15 ATCC Cat#: CCL-33 NIH/3T3 ATCC Cat#: CRL-1658 RIG-I KO A549 This study N/A Syk KO A549 This study N/A IFNAR1 KO A549 This study N/A MDCK ATCC Cat#: CRL-2935 BMDM This study N/A AEC This study N/A Experimental models: organisms/strains Mouse: RIG-I / From Prof. Zhugang Wang, Shanghai Jiao Tong University N/A Mouse: IFNLR1+/ Cyagen Co., Ltd, Guangzhou Cat#: KOCMP-242700-Ifnlr1-B6N Mouse: IFNAR1 / From Prof. Dan Portnoy, University of California N/A Mouse: IFNAR1 / IFNLR1 / This study N/A Mouse: STAT1+/ From Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences N/A Mouse: STAT1Y701F This study N/A Mouse: Sykflox/flox Jackson Laboratory Cat#: 017309 Mouse: Ubc-CreERT2 Shanghai Research Center of Southern Model Organisms N/A Mouse: Sftpc-IRES-iCre (iCre) GemPharmatech Co., Ltd N/A Oligonucleotides shRNA, sgRNA, and siRNA oligonucleotides are in Table S1 N/A N/A Recombinant DNA pSpCas9(BB)-2A-GFP (PX458) From Constance Ciaudo Addgene Plasmid #48138 Syk-PX458 This study N/A RIG-I-PX458 This study N/A IFNAR1-PX458 This study N/A pNL-Myc-tagged MAVS This study N/A pNL-Flag-tagged SykWT This study N/A pNL-Flag-tagged SykSH2 This study N/A pNL-Flag-tagged Sykkinase This study N/A Software and algorithms ImageJ NIH https://imagej.net/ImageJ DNAMAN Lynnon Biosoft N/A Cell Reports 34, 108627, January 19, 2021 e2

Techniques: Activation Assay, Infection, Knockdown, Western Blot, Knock-Out, Control, Stable Transfection, Expressing, Plasmid Preparation

Journal: Cell reports

Article Title: Critical role of Syk-dependent STAT1 activation in innate antiviral immunity.

doi: 10.1016/j.celrep.2020.108627

Figure Lengend Snippet: Figure 5. RIG-I/MAVS-Syk signaling induces the activation of STAT1 during early IAV infection (A) Syk phosphorylation (p-Syk) in WSN-infected WT or RIG-I KO A549 cells was analyzed by WB. (B) p-Syk in lungs of WSN-infected indicated mice was analyzed, as shown in (A). (C) FLAG-Syk-transfected 293T cells were infected with WSN for 3 h. WCLs were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. Shown are representative data from three independent experiments. (D) Diagram of the Syk protein and its truncations used in the co-IP experiments, including Syk, SykSH2-SH2 (Syk-S1), and Sykkinase (Syk-S2). (E) myc-MAVS were cotransfected with Syk or the truncations in 293T cells for 40 h. Cell lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with indicated antibodies. (F) Levels of ISGs in CA04- or PR8-infected indicated cells were determined by quantitative real-time PCR. Data are indicated as means ± SD from three in- dependent experiments. **p < 0.01. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trizol Invitrogen Cat#: 15596018 plasmids expressing myristoylated kinases Addgene, originally fromWilliam Hahn and Jean Zhao Cat#: 1000000012 Critical commercial assays mouse IFN-a ELISA kit eBioscience Cat#: BMS6027 Mouse IFN-b ELISA kit BioLegend Cat#: 439407 Experimental models: cell lines 293T ATCC Cat#: CRL-11268 A549 ATCC Cat#: CCL-185 PK-15 ATCC Cat#: CCL-33 NIH/3T3 ATCC Cat#: CRL-1658 RIG-I KO A549 This study N/A Syk KO A549 This study N/A IFNAR1 KO A549 This study N/A MDCK ATCC Cat#: CRL-2935 BMDM This study N/A AEC This study N/A Experimental models: organisms/strains Mouse: RIG-I / From Prof. Zhugang Wang, Shanghai Jiao Tong University N/A Mouse: IFNLR1+/ Cyagen Co., Ltd, Guangzhou Cat#: KOCMP-242700-Ifnlr1-B6N Mouse: IFNAR1 / From Prof. Dan Portnoy, University of California N/A Mouse: IFNAR1 / IFNLR1 / This study N/A Mouse: STAT1+/ From Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences N/A Mouse: STAT1Y701F This study N/A Mouse: Sykflox/flox Jackson Laboratory Cat#: 017309 Mouse: Ubc-CreERT2 Shanghai Research Center of Southern Model Organisms N/A Mouse: Sftpc-IRES-iCre (iCre) GemPharmatech Co., Ltd N/A Oligonucleotides shRNA, sgRNA, and siRNA oligonucleotides are in Table S1 N/A N/A Recombinant DNA pSpCas9(BB)-2A-GFP (PX458) From Constance Ciaudo Addgene Plasmid #48138 Syk-PX458 This study N/A RIG-I-PX458 This study N/A IFNAR1-PX458 This study N/A pNL-Myc-tagged MAVS This study N/A pNL-Flag-tagged SykWT This study N/A pNL-Flag-tagged SykSH2 This study N/A pNL-Flag-tagged Sykkinase This study N/A Software and algorithms ImageJ NIH https://imagej.net/ImageJ DNAMAN Lynnon Biosoft N/A Cell Reports 34, 108627, January 19, 2021 e2

Techniques: Activation Assay, Infection, Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Real-time Polymerase Chain Reaction