Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a LTM-dependent degradation of viral NS1 protein. Western blot analysis shows reduced NS1 protein levels in cells expressing NS1-N LTM compared with the N LTM mutant , while NS1 mRNA levels remain comparable ( n = 3). b Lysosome dependence of LTM-mediated NS1 degradation. NS1-N LTM protein degradation is blocked by lysosomal inhibitors but not by proteasome or autophagy inhibition. HEK293T cells expressing either NS1-N LTM (left) or NS1-N LTM mutant (right) protein were cultured with or without the proteasome inhibitor MG-132 (10 µM), the autophagy inhibitor 3-methyladenine (3-MA; 10 mM), bafilomycin A1 (Baf A1; 0.4 µM), or chloroquine (CQ; 50 µM) for 6 h. The viral NS1 protein was detected by Western blotting ( n = 3). c Co-immunoprecipitation demonstrating interaction of HSC70 with NS1-N LTM but not with the NS1 LTM mutant ( n = 3). d Dependence of LTM-mediated NS1 degradation on LAMP2A. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing NS1-N LTM (left) or NS1-N LTM mutant (right) protein and collected 24 h post-transfection. Viral NS1 protein was detected by Western blotting ( n = 3). Immunofluorescence analysis showing colocalization of NS1-N LTM with HSC70 ( e ) and LAMP2A ( f ), but not of the NS1-N LTM mutant . Green, NS1; red, HSC70 or LAMP2A; blue, nuclei; yellow, colocalization sites; scale bar, 5 µm. Representative images of at least three independent experiments are shown. LAMP2A-dependent degradation of NS1-N LTM during viral infection in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Replication competence of NS1-N LTM and NS1-N LTM mutant viruses in conventional and LAMP2A-KO HEK293T ( i ) and A549 ( j ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of NS1-N LTM or NS1-N LTM mutant virus in conventional and LAMP2A-KO cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( i ) and ( j ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies were anti-NS1 antibody (GeneTex, Cat# GTX125990, 1:2000 dilution), anti-PA antibody (GeneTex, Cat# GTX118991, 1:2000 dilution), anti-NP antibody (GeneTex, Cat# GTX125989, 1:5000 dilution), anti-M1 antibody (Sino Biological, Cat# 40010-RP01, 1:1000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution).

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Cell Culture, Immunoprecipitation, Transfection, Construct, Immunofluorescence, Infection, Staining, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a Multi-cycle replication kinetics of the indicated viruses in conventional and LAMP2A-KO MDCK cells. Data are presented as means ± s.d ( n = 3). b triLTM-dependent degradation of viral proteins. Western blot analysis shows reduced levels of triLTM-tagged viral proteins compared with mutated triLTM-tagged controls, while corresponding mRNA levels remain comparable ( n = 3). c Lysosome dependence of triLTM-mediated viral protein degradation. HEK293T cells expressing triLTM-tagged or mutated triLTM-tagged viral proteins were cultured in the presence or absence of Baf A1 (0.4 µM) for 6 h and collected for detection of indicated viral proteins by Western blotting ( n = 3). d Co-immunoprecipitation demonstrating interaction of HSC70 with triLTM-tagged viral proteins but not with mutated triLTM-tagged viral proteins ( n = 3). LAMP2A-dependent degradation of triLTM-tagged viral proteins during viral infection in conventional and LAMP2A-KO HEK293T ( e ) and A549 ( f ) cells ( n = 3). Conventional cells and LAMP2A-KO cells were infected with LYTAR 2.0 dual triLTMs or LYTAR 2.0 dual triLTMs mutant virus and collected at 48 h after infection for detection of indicated proteins by Western blotting ( n = 3). Replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant viruses in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant virus in conventional and LAMP2A-KO HEK293T cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( g ) and ( h ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies were anti-NS1 antibody (GeneTex, Cat# GTX125990, 1:2000 dilution), anti-PA antibody (GeneTex, Cat# GTX118991, 1:2000 dilution), anti-NP antibody (GeneTex, Cat# GTX125989, 1:5000 dilution), anti-M1 antibody (Sino Biological, Cat# 40010-RP01, 1:1000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution).

Techniques: Western Blot, Expressing, Cell Culture, Immunoprecipitation, Infection, Mutagenesis, Virus, Immunofluorescence, Staining, Two Tailed Test

Journal: Experimental and Therapeutic Medicine

Article Title: IL-32γ promotes integrin αvβ6 expression through the activation of NF-κB in HSCs

doi: 10.3892/etm.2017.4956

Figure Lengend Snippet: Effect of different concentrations (0, 5, 10 and 20 ng/ml) of IL-32γ on LX-2 activation phenotypes. (A) Growth curves of LX-2 demonstrated that upregulation of IL-32γ promoted proliferation of LX-2. (B) Reverse transcription-quantitative polymerase chain reaction assessing mRNA levels of α-SMA, (C) collagen I, (D) TIMP1, (E) MMP2 and (F) MMP9, representing the activation level of LX-2. (G) Western blot analysis was used to measure collagen I, MMP9, MMP2, α-SMA, TIMP1 and GAPDH expression in whole-cell extracts. Data are presented as the mean ± standard deviation of three experiments. *P<0.01 and **P<0.05 vs. 0 ng/ml IL-32γ. IL-32γ, interleukin-32γ; α-SMA, α-smooth muscle actin; TIMP1, tissue inhibitor of metalloproteinase 1; MMP, matrix metalloproteinases; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Primary antibodies were as follows: Anti-integrin αVβ6 (cat. no. ab97588; Abcam, Cambridge, MA, USA), anti-GAPDH (cat. no. KGAA002-2; Nanjing KeyGen Biotech Co., Ltd.), anti-α-SMA (cat. no. G6669; Sigma-Aldrich; Merck KGaA), collagen type I antibody (cat. no. 600-402-103; Rockland, Limerick, PA, USA), TIMP1 (cat. no. 8946), MMP2 (cat. no. 87809), MMP9 (cat. no. 13667), NF-κB: p65 (cat. no. 8242), p50 (cat. no. 3035) (all Cell Signaling Technology, Inc., Danvers, MA, USA) and IL-32γ (cat. no. 513501; Biolegend, Inc.).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation, Quantitative RT-PCR