a549 cells  (Novus Biologicals)

Journal: bioRxiv

Article Title: The antiviral GTPase MxB is packaged into virions and binds via its N-terminal domain to alphaherpesvirus capsids

doi: 10.1101/2025.08.27.672568

Figure Lengend Snippet: ( a ) Amino acid residue alignment of human MxA(1-43) (Genebank accession number P20591 ), MxB(1-91) and MxB(26-91) ( P20592 ) NTD sequences using the Geneious Alignment with the Blosum62 matrix. Residues were color-coded according to their biochemical properties. ( b ) Domain organization of MxA-NTD (green), MxB(25-715)-NTD (blue), and MxB(1-715)-NTD (red) as well as the common bundling signaling elements (B), the GTPase domains, and the stalks with the L4 loop domains. ( c ) Immunoblot of A549 cells expressing different Mx proteins or control cells using panMx-specific antibodies (M143) and MxA-specific or MxB-specific polyclonal antisera. ( d and e ) A549 cells expressing MxB(1-715) (red), MxB(26-715) (blue), MxA(- 6612) (green) or untransduced cells (black) were infected with HSV-1 at an MOI of 0.001 (d) or with PrV at an MOI of 0.0001 (e). The titers of the culture supernatants were determined by plaque assay. Error bars represent the SD from independent biological replicates (4 for HSV-1; 6 for PrV). The dotted lines indicate the detection limit. Two-way ANOVA tests comparing the log-transformed titers of cells expressing Mx proteins to the untransduced cells. Dunnett: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, nonsignificant. ( f ) A549 cells were infected with PrV at an MOI of 0.0001 and harvested at the indicated hpi. The cell lysates were analyzed by immunoblot using M143 antibodies and antibodies recognizing the PrV capsid (UL19), glycoprotein (gB), tegument (UL37) or non-structural protein (UL31) proteins. The host protein α-actin was used as a loading control. Molecular weight markers are indicated on the left in kDa.

Article Snippet: A549 cells were cultured for 24 h in 6-well plates and treated for 24 h with 1,000 U/mL of human IFN-α2 (NBP2-34971, Novus Biologicals, Bio-Techne GMBH, Wiesbaden, Germany), or infected with HSV-1 at a MOI of 0.01 for 24 or 48 h. The cells were washed with PBS, and total RNA was extracted using 350 μL/well RA1 buffer supplemented with 3.5 μL β-mercaptoethanol, isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany; REF 740955.50), and eluted in 60 μL/well H 2 O. cDNAs were synthesized from 1,000 ng RNA in 12 μL H 2 O using the QuantiTect Reverse Transcription Kit (Qiagen, Germany; Cat. No. 205311) with the reverse transcription step extended to 30 minutes at 42°C, and diluted in Milli-Q water to 100 μL.

Techniques: Residue, Western Blot, Expressing, Control, Infection, Plaque Assay, Transformation Assay, Molecular Weight

Journal: bioRxiv

Article Title: The antiviral GTPase MxB is packaged into virions and binds via its N-terminal domain to alphaherpesvirus capsids

doi: 10.1101/2025.08.27.672568

Figure Lengend Snippet: A549 cells were mock-treated (control), treated with human IFN-α2 at 1000 U/mL for 24 h, or infected with HSV-1 at an MOI of 0.01 for 24 or 48 h. ( a - e, g, h ) The expression of HSV1-ICP0 (infected cell protein 0), and host IFN-β (interferon beta), IRF7 (interferon-response factor), Mx1, Mx2, ISG-15 (interferon-stimulated gene 15), and IL-6 (interleukin 6) were quantified relative to α-actin by the 2 −ΔCT method. Means ± SD of triplicates with each dot representing the mean of technical duplicates. One-way ANOVA test with Dunnett multiple comparison: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. ( f ) The proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with panMx- specific antibody (M143), with polyclonal sera specific for MxA or MxB, or with a polyclonal rabbit serum detecting the major HSV1 capsid protein VP5. Actin was used as a loading control. Molecular weight markers are indicated on the left.

Article Snippet: A549 cells were cultured for 24 h in 6-well plates and treated for 24 h with 1,000 U/mL of human IFN-α2 (NBP2-34971, Novus Biologicals, Bio-Techne GMBH, Wiesbaden, Germany), or infected with HSV-1 at a MOI of 0.01 for 24 or 48 h. The cells were washed with PBS, and total RNA was extracted using 350 μL/well RA1 buffer supplemented with 3.5 μL β-mercaptoethanol, isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany; REF 740955.50), and eluted in 60 μL/well H 2 O. cDNAs were synthesized from 1,000 ng RNA in 12 μL H 2 O using the QuantiTect Reverse Transcription Kit (Qiagen, Germany; Cat. No. 205311) with the reverse transcription step extended to 30 minutes at 42°C, and diluted in Milli-Q water to 100 μL.

Techniques: Control, Infection, Expressing, Comparison, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: The antiviral GTPase MxB is packaged into virions and binds via its N-terminal domain to alphaherpesvirus capsids

doi: 10.1101/2025.08.27.672568

Figure Lengend Snippet: Protein composition of L-particle and H particles harvested from infected A-549-MxA(1-662) or A549-MxB(1-715) cells. (a, b) Relative protein quantification of H-particles secreted from (a) MxA(1-662)-A5439 or (b) A549-MxB(1-715) expressing cells compared to the respective whole cell lysates. Several categories of viral and host proteins have been color-coded, in addition to the highlighted Mx proteins (red), KIF5B kinesin-1 isoform (black), and HSV1- VP5 major capsid protein (black). Means of fold-change differences and P values of a two- sided t-tests without multiple hypothesis correction were based on quadruplicate measurements for cell lysates and triplicate measurements for H particles. Quantification was performed using the LFQ algorithm in MaxQuant, and only unique peptides were used. (c, d) Absolute quantification of the proteins of H-particles secreted from (c) MxA(1-662)-A5439 or (d) A549-MxB(1-715) expressing cells compared to the respective whole cell lysates based on the intensity-based absolute quantification (iBAQ)-values that were summed up across three replicates, compared to enrichment levels. (e) Comparison of the log2-transformed protein intensity (LFQ) of the proteins of H particles secreted from MxA(1-662)-A5439 or A549-MxB(1-715) expressing cells compared to the respective whole cell lysates with individual dots indicating all replicates and the lines indicating the respective mean values. The p-values are based on two-sided t-tests. ( f ) Control, MxA or MxB overexpressing A549 cells were left untreated or infected with HSV-1 at an MOI of 0.001 for 48 or 54h. Immunoblot of total cell lysates probed with the panMx-specific antibodies (M143), with MxA- or MxB-specific polyclonal antisera, or with HSV1-VP5 antibodies. Actin was used as a loading control. Molecular weight markers are indicated on the left in kDa. (g, h) The iBAQ-values for the protein compositions of L- and H-particles secreted from HSV-1 infected (g) MxA(1-662)-A5439 or (h) A549-MxB(1-715) expressing cells were analysed for all biological replicates. The color code indicates different categories of viral and host proteins.

Article Snippet: A549 cells were cultured for 24 h in 6-well plates and treated for 24 h with 1,000 U/mL of human IFN-α2 (NBP2-34971, Novus Biologicals, Bio-Techne GMBH, Wiesbaden, Germany), or infected with HSV-1 at a MOI of 0.01 for 24 or 48 h. The cells were washed with PBS, and total RNA was extracted using 350 μL/well RA1 buffer supplemented with 3.5 μL β-mercaptoethanol, isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany; REF 740955.50), and eluted in 60 μL/well H 2 O. cDNAs were synthesized from 1,000 ng RNA in 12 μL H 2 O using the QuantiTect Reverse Transcription Kit (Qiagen, Germany; Cat. No. 205311) with the reverse transcription step extended to 30 minutes at 42°C, and diluted in Milli-Q water to 100 μL.

Techniques: Infection, Expressing, Quantitative Proteomics, Comparison, Transformation Assay, Control, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: The antiviral GTPase MxB is packaged into virions and binds via its N-terminal domain to alphaherpesvirus capsids

doi: 10.1101/2025.08.27.672568

Figure Lengend Snippet: ( a ) Workflow of the particle fractionation and capsid preparation: Extracellular particles were harvested from A549 cells expressing MxA(1-662), MxB(26-715), or MxB(1-715) and infected with HSV-1 or PrV resuspended in an equal volume of PBS (1), and trypsin was added to digest any proteins attached to the particle surfaces, followed by the addition of trypsin inhibitor (2). The particles were kept in PBS (3) or lysed with 1% TX-100 and 0.5 M KCl (4). The lysates were layered on top of a 30% sucrose cushion in PBS, and centrifuged to sediment the capsids and capsid-associated proteins (5) or treated with 1% TX-100 at 0.5 M KCl (6). Samples from each experiment and each preparation step were resuspended in SDS sample buffer. Created with BioRender.com. ( b and c ) HSV-1 particle fractions containing 2 × 10 8 pfu (b) or PrV (c) particles containing 4 × 10 7 pfu (c) were used as starting material (1). The Western blot analyses show (panel 1) the initial purified, extracellular particles, (2) the trypsin-treated particles, the PBS-treated particles (3) before and (5) after UC, and the Triton/KCl-treated particles (4) before and (6) after UC. The pan-Mx-specific (M143) antibody and antibodies specific for HSV-1 and PrV proteins, were used for detection of capsid (VP5 and pUL19), glycoproteins (gD and gB) and tegument proteins (pUL36, pUL37, VP22, pUL11 and UL37, UL31). ( d and e ) MxB(1-715) is protected from trypsin digestion in the viral particles. Purified extracellular particles of PrV (4 × 10 7 pfu, as described in panel c) were (d1) mock-treated or (e1) treated with 1% Triton X-100 in PBS. Then the mock-treated (d2) and Triton-treated (e2) particles were incubated with trypsin for 30 min at 37°C. The digest was stopped by addition of trypsin inhibitor. Finally, the mock- or trypsin-treated particle fractions were ultracentrifuged and the resulting pellets of the mock/trypsin (d3) or Triton/trypsin (e3)-treatments were resuspended in SDS sample buffer. Each step of particle treatment and the final UC pellets were analyzed by Western blot as described in panel c. The panels show representative results of (b) three and (c, d, e) two independent experiments.

Article Snippet: A549 cells were cultured for 24 h in 6-well plates and treated for 24 h with 1,000 U/mL of human IFN-α2 (NBP2-34971, Novus Biologicals, Bio-Techne GMBH, Wiesbaden, Germany), or infected with HSV-1 at a MOI of 0.01 for 24 or 48 h. The cells were washed with PBS, and total RNA was extracted using 350 μL/well RA1 buffer supplemented with 3.5 μL β-mercaptoethanol, isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany; REF 740955.50), and eluted in 60 μL/well H 2 O. cDNAs were synthesized from 1,000 ng RNA in 12 μL H 2 O using the QuantiTect Reverse Transcription Kit (Qiagen, Germany; Cat. No. 205311) with the reverse transcription step extended to 30 minutes at 42°C, and diluted in Milli-Q water to 100 μL.

Techniques: Fractionation, Expressing, Infection, Western Blot, Purification, Incubation

Journal: bioRxiv

Article Title: The antiviral GTPase MxB is packaged into virions and binds via its N-terminal domain to alphaherpesvirus capsids

doi: 10.1101/2025.08.27.672568

Figure Lengend Snippet: ( a to c ) Extracellular particles were harvested from HSV-1 infected A549 cells expressing MxA(1-662), MxB(26- 715) or MxB(1-715), resuspended in PBS, treated with trypsin and absorbed on EM grids. The samples were left untreated in PBS (a; PBS->PBS), or the viral envelope was opened by osmotic lysis in pure H 2 O. Then the lysed particles were incubated in PBS (b; H 2 O > PBS) or with 0.5 M KCl (c; H 2 O > high salt) for 30 min at RT. All specimens were processed for immunogold labeling analysis using the pan-Mx-specific M143 antibody. Scale bars represent 50 nm. ( d ) The number of protein A gold particles within a distance of 25 nm distance to 100 virons were counted in three technical replicates, and the mean number of gold particles per virion were calculated and statistically evaluated using a 2-way ANOVA test.

Article Snippet: A549 cells were cultured for 24 h in 6-well plates and treated for 24 h with 1,000 U/mL of human IFN-α2 (NBP2-34971, Novus Biologicals, Bio-Techne GMBH, Wiesbaden, Germany), or infected with HSV-1 at a MOI of 0.01 for 24 or 48 h. The cells were washed with PBS, and total RNA was extracted using 350 μL/well RA1 buffer supplemented with 3.5 μL β-mercaptoethanol, isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany; REF 740955.50), and eluted in 60 μL/well H 2 O. cDNAs were synthesized from 1,000 ng RNA in 12 μL H 2 O using the QuantiTect Reverse Transcription Kit (Qiagen, Germany; Cat. No. 205311) with the reverse transcription step extended to 30 minutes at 42°C, and diluted in Milli-Q water to 100 μL.

Techniques: Infection, Expressing, Lysis, Incubation, Labeling

Journal: Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/jci186863

Figure Lengend Snippet: Figure 2. Overexpression of NECTIN2 in LUAD and tumor cell sialylation. (A and B) In silico analysis indicated that expression of NECTIN2 is significantly overexpressed and associated with poor survival outcomes in both early- and late-stage LUAD patients. (C) Protein-protein interaction networking analysis reveals that tumor cells expressing NECTIN2 induce T cell dysfunction through TIGIT binding, which is associated with immune suppression pathways. (D) IHC analysis shows the overexpression of NECTIN2 in LUAD. Data were analyzed using 2-tailed t test (n = 38). Original magnification, ×10. (E) The expression of MUC5AC and NECTIN2 drastically decreased in ST6GalNAc-I–KO and MUC5AC-KD cells (A549 and H1437). (F and G) Immunoprecipitation assay shows NECTIN2 sialylation in LUAD cells. (H) SNA pull-down was performed on A549 cell lysates, followed by immunoblotting with NECTIN2 antibody, suggesting that NECTIN2 carries STn in LUAD cells. (I) Immunofluorescence assay reveals the decreased association of NECTIN2 and STn in A549 ST6GalNAc-I–KO cells, and the bar diagram represents the quantification of NECTIN2 and STn using arith- metic mean intensity. Data were analyzed using 2-tailed t test (n = 3). Scale bars: 5 μm.

Article Snippet: MUC5AC (CLH2 clone), PRRC1, and IgG antibodies were immunoprecipitated with total lysates (500 μg) isolated from A549 cells using protein A+G Sepharose beads (catalog sc-2003, Santa Cruz Biotechnology).

Techniques: Over Expression, In Silico, Expressing, Binding Assay, Immunoprecipitation, Western Blot, Immunofluorescence

Journal: Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/jci186863

Figure Lengend Snippet: Figure 5. VCAN is the common target of ST6GalNAc-I/MUC5AC in LUAD. (A and B) Volcano plots for the transcriptomic data (RNA-Seq) [FDR < 0.05 and log2(fold change) ≤ 1] and proteomic data [FDR < 0.05 and log2(fold change) ≤ 1] reveal that MUC5AC regulates VCAN in LUAD. Red represents significantly upregulated genes/proteins, and blue represents significantly downregulated genes/proteins. (C) VCAN is a common downstream molecule of MUC5AC and ST6GalNAc-I as demonstrated by a Venn diagram using MS data (both ST6GalNAc-I–KO and MUC5AC-KD). (D) Overall survival analysis indicated that the high expression of VCAN showed a poor prognosis in LUAD. (E and F) Immunoblots show that VCAN and its isoform VCAN-V1 are drastically decreased along with MUC5AC in ST6GalNAc-I–KO and MUC5AC-KD cells (A549 and H1437). (G) Confocal images show that MUC5AC and proteoglycan VCAN-V1 are strongly colocalized in the extracellular tumor matrix region of MUC5AC and VCAN-V1 in LUAD cells. Scale bars: 5 μm and 10 μm. Significance was determined by 2-tailed t test (n = 3). Scr, scramble. (H) Immunoprecipitation of MUC5AC in A549 cell lysates, followed by blotting with SNA lectin. The arrow shows SNA staining in the MUC5AC region. (I) SNA pull-down in A549 cell lysates and blotting with MUC5AC, suggesting that MUC5AC carries STn in LUAD cells.

Article Snippet: MUC5AC (CLH2 clone), PRRC1, and IgG antibodies were immunoprecipitated with total lysates (500 μg) isolated from A549 cells using protein A+G Sepharose beads (catalog sc-2003, Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Expressing, Western Blot, Immunoprecipitation, Staining

Journal: Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/jci186863

Figure Lengend Snippet: Figure 7. MUC5AC interacts with PRRC1 in LUAD. (A) MS-based cellular pathway analysis indicated that MUC5AC is associated with Golgi-associated vesicle and COPI-coated vesicle processes. (B) Experimental design for MS-based interactome studies. MS analysis was performed using A549 MUC5AC-KD and scramble cells. (C) MS-based MUC5AC interactome studies revealed that MUC5AC strongly interacts with PRRC1 as represented by heatmap. (D and E) Reciprocal immunoprecipitation assays also revealed that MUC5AC strongly interacts with PRRC1 in LUAD cells. (F and G) Further, confocal images show that MUC5AC and PRRC1 were strongly colocalized in the Golgi region along with the Golgi-specific marker GM130. Significance was determined by 2-tailed t test (n = 3). Scale bars: 5 μm and 10 μm. (H) Schematic diagram shows the MUC5AC glycosylation process in LUAD cells.

Article Snippet: MUC5AC (CLH2 clone), PRRC1, and IgG antibodies were immunoprecipitated with total lysates (500 μg) isolated from A549 cells using protein A+G Sepharose beads (catalog sc-2003, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Marker, Glycoproteomics

Journal: Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/jci186863

Figure Lengend Snippet: Figure 8. MUC5AC is associated with angiogenesis and tumor development. (A) Scheme of in vivo intratracheal models using human non–small cell lung cancer cells in athymic nude mice. (B) Mice injected with A549 MUC5AC-KD cells had significantly reduced lung tumor foci compared with mice inject- ed with respective control cells (n = 8). (B and C) IHC shows that the expression of Ki-67, MUC5AC, CD31, VCAN, and VCAN-V1 was significantly reduced in MUC5AC-KD tumors. Original magnification, ×10 for H&E and ×40 for IHC. Significance was determined by 2-tailed t test (n = 3). (C, top panel) The microvessel density was quantified using CD31-stained tumor sections in 8 fields for each tumor section and presented as a mean number per field of view (FOV; 0.2 mm2). Significance was determined by 2-tailed t test (n = 3). (D and E) Confocal images show decreased colocalization of CD31 with MUC5AC and VCAN-V1 in MUC5AC KD–derived xenograft. Scale bars: 10 μm.

Article Snippet: MUC5AC (CLH2 clone), PRRC1, and IgG antibodies were immunoprecipitated with total lysates (500 μg) isolated from A549 cells using protein A+G Sepharose beads (catalog sc-2003, Santa Cruz Biotechnology).

Techniques: In Vivo, Injection, Control, Expressing, Staining, Derivative Assay