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Santa Cruz Biotechnology stx12 shrna human lentivirus

<t>STX12,</t> eIF4EBP1, and eIF4EBP2 are direct targets of miR-31 (A) The differentially expressed mRNAs among HCT116 cells transfected with Lv-miR-31, Lv-NC, Lv-anti-miR-31, or Lv-anti-NC, as identified by microarray. (B) eIF4EBP1/2 and STX12 were identified as the direct targets of miR-31 as detected and verified by the TargetScan prediction, MiRnada, miRDB, GO annotation, and target of microarray, respectively. (C) The region of the human eIF4EBP1/2 and STX12 mRNA 3′UTR was predicted to be targeted by miR-31 (TargetScan 7.2). (D) Luciferase reporter plasmids were generated with either WT or MT miR-31 binding sites in 3′-UTR of eIF4EBP1/2 and STX12. The activity of the luciferase constructs was evaluated and normalized to the activity of Renilla luciferase after transfecting HEK293T cells with a miR-31 mimic, an inhibitor, or a control. (E and F) HCT116 cells were transfected with the mimic or the inhibitor of miR-31. After co-culture with F. nucleatum for 4 h, the mRNA and protein expression of eIF4EBP1/2 and STX12 was detected by RT-qPCR and Western blotting, respectively.

Stx12 Shrna Human Lentivirus, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis"

Article Title: MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis

Journal: iScience

doi: 10.1016/j.isci.2023.106770

STX12, eIF4EBP1, and eIF4EBP2 are direct targets of miR-31 (A) The differentially expressed mRNAs among HCT116 cells transfected with Lv-miR-31, Lv-NC, Lv-anti-miR-31, or Lv-anti-NC, as identified by microarray. (B) eIF4EBP1/2 and STX12 were identified as the direct targets of miR-31 as detected and verified by the TargetScan prediction, MiRnada, miRDB, GO annotation, and target of microarray, respectively. (C) The region of the human eIF4EBP1/2 and STX12 mRNA 3′UTR was predicted to be targeted by miR-31 (TargetScan 7.2). (D) Luciferase reporter plasmids were generated with either WT or MT miR-31 binding sites in 3′-UTR of eIF4EBP1/2 and STX12. The activity of the luciferase constructs was evaluated and normalized to the activity of Renilla luciferase after transfecting HEK293T cells with a miR-31 mimic, an inhibitor, or a control. (E and F) HCT116 cells were transfected with the mimic or the inhibitor of miR-31. After co-culture with F. nucleatum for 4 h, the mRNA and protein expression of eIF4EBP1/2 and STX12 was detected by RT-qPCR and Western blotting, respectively.

Figure Legend Snippet: STX12, eIF4EBP1, and eIF4EBP2 are direct targets of miR-31 (A) The differentially expressed mRNAs among HCT116 cells transfected with Lv-miR-31, Lv-NC, Lv-anti-miR-31, or Lv-anti-NC, as identified by microarray. (B) eIF4EBP1/2 and STX12 were identified as the direct targets of miR-31 as detected and verified by the TargetScan prediction, MiRnada, miRDB, GO annotation, and target of microarray, respectively. (C) The region of the human eIF4EBP1/2 and STX12 mRNA 3′UTR was predicted to be targeted by miR-31 (TargetScan 7.2). (D) Luciferase reporter plasmids were generated with either WT or MT miR-31 binding sites in 3′-UTR of eIF4EBP1/2 and STX12. The activity of the luciferase constructs was evaluated and normalized to the activity of Renilla luciferase after transfecting HEK293T cells with a miR-31 mimic, an inhibitor, or a control. (E and F) HCT116 cells were transfected with the mimic or the inhibitor of miR-31. After co-culture with F. nucleatum for 4 h, the mRNA and protein expression of eIF4EBP1/2 and STX12 was detected by RT-qPCR and Western blotting, respectively.

Techniques Used: Transfection, Microarray, Luciferase, Generated, Binding Assay, Activity Assay, Construct, Control, Co-Culture Assay, Expressing, Quantitative RT-PCR, Western Blot

miR-31 inhibits STX12 to block the fusion of autophagosomes with lysosomes during Fusobacterium nucleatum infection (A) The protein expression of STX12 in miR-31 overexpressed or inhibited HCT116 cells infected with F. nucleatum (MOI = 100:1) for 6 h. (B) Western blotting detected the protein expression of STX12 in F. nucleatum -infected miR-31 wt or miR-31 −/− mice tissues (n = 5 per group). (C) The protein expression of STX12 was detected by Western blotting in F. nucleatum- positive CRC tissues (Fn + CRC, n = 5) and F. nucleatum- negative CRC tissues (Fn − CRC, n = 5). (D and E) HCT116 cells were treated with Lv-sh-NC or Lv-sh-STX12, and then infected with F. nucleatum (MOI = 100). The expression of MAP1LC3B-I/II and SQSTM1 was detected by Western blotting, and the mRFP-EGFP-LC3 fusion protein was detected by laser scanning confocal microscopy (LSCM). (F) After pretreatment with Lv-sh-NC or Lv-sh-STX12, HCT116 cells were infected with F. nucleatum (MOI = 100:1) for 6 h. Then the intracellular survival of F. nucleatum in HCT116 cells were detected by TEM. (G) HCT116 cells treated with Lv-sh-STX12 were transiently transfected with a miR-31 mimic or an inhibitor, and then infected with F. nucleatum, the intracellular survival of F. nucleatum was detected 6 h post-infection. (H) Both miR-31 wt and miR-31 −/− mice were initially injected with Av-sh-NC or Av-sh-STX12 via the caudal vein and then infected with F. nucleatum for 20 weeks. (I) Representative images of H&E staining of the colonic epithelium from miR-31 wt and miR-31 −/− mice. (J) The colonization of F. nucleatum in colorectal tissues of miR-31 wt and miR-31 −/− mice. ∗p < 0.05.

Figure Legend Snippet: miR-31 inhibits STX12 to block the fusion of autophagosomes with lysosomes during Fusobacterium nucleatum infection (A) The protein expression of STX12 in miR-31 overexpressed or inhibited HCT116 cells infected with F. nucleatum (MOI = 100:1) for 6 h. (B) Western blotting detected the protein expression of STX12 in F. nucleatum -infected miR-31 wt or miR-31 −/− mice tissues (n = 5 per group). (C) The protein expression of STX12 was detected by Western blotting in F. nucleatum- positive CRC tissues (Fn + CRC, n = 5) and F. nucleatum- negative CRC tissues (Fn − CRC, n = 5). (D and E) HCT116 cells were treated with Lv-sh-NC or Lv-sh-STX12, and then infected with F. nucleatum (MOI = 100). The expression of MAP1LC3B-I/II and SQSTM1 was detected by Western blotting, and the mRFP-EGFP-LC3 fusion protein was detected by laser scanning confocal microscopy (LSCM). (F) After pretreatment with Lv-sh-NC or Lv-sh-STX12, HCT116 cells were infected with F. nucleatum (MOI = 100:1) for 6 h. Then the intracellular survival of F. nucleatum in HCT116 cells were detected by TEM. (G) HCT116 cells treated with Lv-sh-STX12 were transiently transfected with a miR-31 mimic or an inhibitor, and then infected with F. nucleatum, the intracellular survival of F. nucleatum was detected 6 h post-infection. (H) Both miR-31 wt and miR-31 −/− mice were initially injected with Av-sh-NC or Av-sh-STX12 via the caudal vein and then infected with F. nucleatum for 20 weeks. (I) Representative images of H&E staining of the colonic epithelium from miR-31 wt and miR-31 −/− mice. (J) The colonization of F. nucleatum in colorectal tissues of miR-31 wt and miR-31 −/− mice. ∗p < 0.05.

Techniques Used: Blocking Assay, Infection, Expressing, Western Blot, Confocal Microscopy, Transfection, Injection, Staining

NF-κB is a transcription factor for miR-31 A) KEGG pathway analysis of a total of 1074 mRNAs involved in F. nucleatum -associated CRC. (B) Western blotting was performed to measure the protein levels of phosphorylated p65 in the cytoplasm and nucleus of HCT116 cells after F. nucleatum infection. (C and D) miR-31 and pri-miR-31 expression were detected by RT-qPCR in HCT116 or LoVo cells transfected with Lv-sh p65. (E) HCT116 cells transfected with Lv-sh p65 were infected with F. nucleatum for 2 h, and the primary transcript (pri-miRNA) of miR-31 was quantified by real-time PCR. ∗p < 0.05. (F) pGL3 luciferase reporter plasmids that either have the wild-type (WT) or mutant (MT) p65 binding site in the miR-31 promoter. (G) Luciferase constructs (pGL3-WT or pGL3-MT) or Lv-sh p65 were co-transfected into HEK293T cells prior to F. nucleatum infection. The activity of luciferase was adjusted to match that of Renilla luciferase. ∗p < 0.05. (H) miR-31 promoter was detected in the chromatin sample immunoprecipitated from HCT116 (left) and LoVo (right) cells using an antibody against p65. A schematic representation of the mechanism by which F. nucleatum induces intestinal inflammation and colorectal tumorigenesis via targeting NF-κB, miR-31, STX12, and the autophagy pathway.

Figure Legend Snippet: NF-κB is a transcription factor for miR-31 A) KEGG pathway analysis of a total of 1074 mRNAs involved in F. nucleatum -associated CRC. (B) Western blotting was performed to measure the protein levels of phosphorylated p65 in the cytoplasm and nucleus of HCT116 cells after F. nucleatum infection. (C and D) miR-31 and pri-miR-31 expression were detected by RT-qPCR in HCT116 or LoVo cells transfected with Lv-sh p65. (E) HCT116 cells transfected with Lv-sh p65 were infected with F. nucleatum for 2 h, and the primary transcript (pri-miRNA) of miR-31 was quantified by real-time PCR. ∗p < 0.05. (F) pGL3 luciferase reporter plasmids that either have the wild-type (WT) or mutant (MT) p65 binding site in the miR-31 promoter. (G) Luciferase constructs (pGL3-WT or pGL3-MT) or Lv-sh p65 were co-transfected into HEK293T cells prior to F. nucleatum infection. The activity of luciferase was adjusted to match that of Renilla luciferase. ∗p < 0.05. (H) miR-31 promoter was detected in the chromatin sample immunoprecipitated from HCT116 (left) and LoVo (right) cells using an antibody against p65. A schematic representation of the mechanism by which F. nucleatum induces intestinal inflammation and colorectal tumorigenesis via targeting NF-κB, miR-31, STX12, and the autophagy pathway.

Techniques Used: Western Blot, Infection, Expressing, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction, Luciferase, Mutagenesis, Binding Assay, Construct, Activity Assay, Immunoprecipitation

Figure Legend Snippet:

Techniques Used: Control, Virus, Luciferase, Plasmid Preparation, shRNA, Recombinant, Protein Extraction, BIA-KA, CCK-8 Assay, Reverse Transcription, Knock-Out, Software, Microscopy

Image Search Results

Protective effects of BoNT/A in R28 cells under hypoxic stress. ( A ) Schematic illustration of the experimental timeline. R28 cells were pretreated with BoNT/A for 2 h, followed by CoCl 2 (200 μM) exposure, and samples were collected at 3 h and 24 h. ( B ) Cell viability was tested using the CCK-8 assay. Data are presented as mean ± SEM (** p < 0.01 vs. control; # p < 0.05 vs. CoCl 2 ). ( C ) Expression levels of target proteins in R28 cells exposed to CoCl 2 for 3 h. ( D ) Immunocytochemistry analyses revealed multiple BoNT/A-related effects. The first panel confirmed BoNT/A activity by cleaved-SNAP25 staining (green). The second panel showed increased SOCS3 expression after BoNT/A treatment. The third panel demonstrated that BoNT/A suppressed the CoCl 2 -induced upregulation of Hv1 and Nox2. The fourth panel revealed that CoCl 2 promoted NF-κB nuclear translocation, which was reduced by BoNT/A pretreatment. The fifth panel showed that Brn3a expression, markedly decreased in CoCl 2 -treated cells, was preserved in the CoCl 2 + BoNT/A group, indicating protection of retinal ganglion cell integrity. Scale bars: 50 μm. ( E ) VDAC1 (red) displayed pronounced perinuclear clustering in CoCl 2 -treated cells, consistent with mitochondrial aggregation under stress, whereas this effect was attenuated by BoNT/A pretreatment (arrows). Syntaxin12 (red), normally extended in filamentous structures, exhibited fragmentation in CoCl 2 + BoNT/A samples at 24 h, suggesting cleavage or disruption of its morphology. The yellow boxes indicate the regions that were magnified and shown below to highlight these structural changes. Scale bars: 50 μm (overview); 25 μm (insets). Statistical significance was indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ### p < 0.001 vs. CoCl 2 .

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Modulation of the Nox2–Hv1–ROS Axis by Botulinum Neurotoxin A Confers Protection Against CoCl 2 -Induced Retinal Hypoxic Injury

doi: 10.3390/ijms262110806

Figure Lengend Snippet: Protective effects of BoNT/A in R28 cells under hypoxic stress. ( A ) Schematic illustration of the experimental timeline. R28 cells were pretreated with BoNT/A for 2 h, followed by CoCl 2 (200 μM) exposure, and samples were collected at 3 h and 24 h. ( B ) Cell viability was tested using the CCK-8 assay. Data are presented as mean ± SEM (** p < 0.01 vs. control; # p < 0.05 vs. CoCl 2 ). ( C ) Expression levels of target proteins in R28 cells exposed to CoCl 2 for 3 h. ( D ) Immunocytochemistry analyses revealed multiple BoNT/A-related effects. The first panel confirmed BoNT/A activity by cleaved-SNAP25 staining (green). The second panel showed increased SOCS3 expression after BoNT/A treatment. The third panel demonstrated that BoNT/A suppressed the CoCl 2 -induced upregulation of Hv1 and Nox2. The fourth panel revealed that CoCl 2 promoted NF-κB nuclear translocation, which was reduced by BoNT/A pretreatment. The fifth panel showed that Brn3a expression, markedly decreased in CoCl 2 -treated cells, was preserved in the CoCl 2 + BoNT/A group, indicating protection of retinal ganglion cell integrity. Scale bars: 50 μm. ( E ) VDAC1 (red) displayed pronounced perinuclear clustering in CoCl 2 -treated cells, consistent with mitochondrial aggregation under stress, whereas this effect was attenuated by BoNT/A pretreatment (arrows). Syntaxin12 (red), normally extended in filamentous structures, exhibited fragmentation in CoCl 2 + BoNT/A samples at 24 h, suggesting cleavage or disruption of its morphology. The yellow boxes indicate the regions that were magnified and shown below to highlight these structural changes. Scale bars: 50 μm (overview); 25 μm (insets). Statistical significance was indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ### p < 0.001 vs. CoCl 2 .

Article Snippet: Syntaxin12 , Rabbit , Proteintech, Rosemont, IL, USA , 14259-1-AP , 1:200 (ICC), 1:1000 (WB).

Techniques: CCK-8 Assay, Control, Expressing, Immunocytochemistry, Activity Assay, Staining, Translocation Assay, Disruption

BoNT/A protects retinal structure and reduces retinal ganglion cell loss in an ex vivo model. ( A ) Experimental scheme of the ex vivo retina model. Rat retinas were cultured ex vivo, pretreated with BoNT/A (2 h), and subsequently exposed to CoCl 2 (300 μM). Samples were collected at 3h, 4D (days), and 8D. ( B ) Representative hematoxylin–eosin (H&E) staining images of paraffin-embedded retinal cross-sections at the indicated time points. Retinal thickness was quantified for the whole retina as well as for specific layers, including the inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL). CoCl 2 treatment caused progressive thinning of the retina and individual layers, whereas BoNT/A pretreatment preserved retinal thickness at levels comparable to controls. Scale bar: 50 μm. ( C ) Immunofluorescence staining of cleaved-SNAP25 confirmed BoNT/A enzymatic activity in retinal tissues. Robust cleaved-SNAP25 signals were observed primarily in the IPL of BoNT/A-pretreated retinas but were absent in control and CoCl 2 -only groups. Enlarged views highlight localization within the IPL and adjacent INL. Quantitative fluorescence analyses are shown in the adjacent graphs. Scale bars: 50 μm (overview); 25 μm (insets). ( D ) Brn3a immunostaining demonstrated that BoNT/A pretreatment preserved retinal ganglion cell labeling compared with CoCl 2 -treated retinas. Scale bar: 50 μm. ( E ) TUNEL assay revealed abundant apoptotic nuclei (green) in CoCl 2 -treated retinas, which were markedly reduced in BoNT/A-pretreated samples, indicating protection against hypoxia-induced apoptosis. Scale bar: 50 μm. ( F ) IBA1 (red) and GFAP (green) were strongly upregulated in CoCl 2 -treated retinas, confirming successful induction of hypoxia, but were reduced by BoNT/A treatment, indicating suppression of glial activation. Scale bar: 50 μm. ( G ) Immunostaining for Hv1 (red) and Nox2 (green) revealed increased expression in CoCl 2 -treated retinas, which was reduced following BoNT/A pretreatment. Scale bar: 50 μm. ( H ) Western blot analyses using the same antibodies as in R28 cells showed that Nox2, Hv1, COX2, NLRP3, and TNF-α were elevated by CoCl 2 and downregulated by BoNT/A. In contrast, SOCS3, GAP43, and Syntaxin12 were increased in BoNT/A-treated samples, suggesting enhanced anti-inflammatory and neuroprotective signaling. Statistical significance was indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CoCl 2 . + p < 0.05, ++ p < 0.01, +++ p < 0.001 BoNT/A 0.25 IU vs. 0.5 IU. Scale bar: 50 μm.

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Modulation of the Nox2–Hv1–ROS Axis by Botulinum Neurotoxin A Confers Protection Against CoCl 2 -Induced Retinal Hypoxic Injury

doi: 10.3390/ijms262110806

Figure Lengend Snippet: BoNT/A protects retinal structure and reduces retinal ganglion cell loss in an ex vivo model. ( A ) Experimental scheme of the ex vivo retina model. Rat retinas were cultured ex vivo, pretreated with BoNT/A (2 h), and subsequently exposed to CoCl 2 (300 μM). Samples were collected at 3h, 4D (days), and 8D. ( B ) Representative hematoxylin–eosin (H&E) staining images of paraffin-embedded retinal cross-sections at the indicated time points. Retinal thickness was quantified for the whole retina as well as for specific layers, including the inner plexiform layer (IPL), inner nuclear layer (INL), and outer nuclear layer (ONL). CoCl 2 treatment caused progressive thinning of the retina and individual layers, whereas BoNT/A pretreatment preserved retinal thickness at levels comparable to controls. Scale bar: 50 μm. ( C ) Immunofluorescence staining of cleaved-SNAP25 confirmed BoNT/A enzymatic activity in retinal tissues. Robust cleaved-SNAP25 signals were observed primarily in the IPL of BoNT/A-pretreated retinas but were absent in control and CoCl 2 -only groups. Enlarged views highlight localization within the IPL and adjacent INL. Quantitative fluorescence analyses are shown in the adjacent graphs. Scale bars: 50 μm (overview); 25 μm (insets). ( D ) Brn3a immunostaining demonstrated that BoNT/A pretreatment preserved retinal ganglion cell labeling compared with CoCl 2 -treated retinas. Scale bar: 50 μm. ( E ) TUNEL assay revealed abundant apoptotic nuclei (green) in CoCl 2 -treated retinas, which were markedly reduced in BoNT/A-pretreated samples, indicating protection against hypoxia-induced apoptosis. Scale bar: 50 μm. ( F ) IBA1 (red) and GFAP (green) were strongly upregulated in CoCl 2 -treated retinas, confirming successful induction of hypoxia, but were reduced by BoNT/A treatment, indicating suppression of glial activation. Scale bar: 50 μm. ( G ) Immunostaining for Hv1 (red) and Nox2 (green) revealed increased expression in CoCl 2 -treated retinas, which was reduced following BoNT/A pretreatment. Scale bar: 50 μm. ( H ) Western blot analyses using the same antibodies as in R28 cells showed that Nox2, Hv1, COX2, NLRP3, and TNF-α were elevated by CoCl 2 and downregulated by BoNT/A. In contrast, SOCS3, GAP43, and Syntaxin12 were increased in BoNT/A-treated samples, suggesting enhanced anti-inflammatory and neuroprotective signaling. Statistical significance was indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CoCl 2 . + p < 0.05, ++ p < 0.01, +++ p < 0.001 BoNT/A 0.25 IU vs. 0.5 IU. Scale bar: 50 μm.

Article Snippet: Syntaxin12 , Rabbit , Proteintech, Rosemont, IL, USA , 14259-1-AP , 1:200 (ICC), 1:1000 (WB).

Techniques: Ex Vivo, Cell Culture, Staining, Immunofluorescence, Activity Assay, Control, Fluorescence, Immunostaining, Labeling, TUNEL Assay, Activation Assay, Expressing, Western Blot

Suppression of the Nox2–Hv1 Axis and Enhancement of Neuroprotective Signaling by BoNT/A in hypoxic injury. Cobalt chloride (CoCl 2 )–induced hypoxia stabilizes HIF-1α and triggers excessive ROS production, leading to metabolic stress within cells. This condition further drives inflammation (Hv1, Nox2, NLRP3, COX2, TNF-α), retinal thinning, apoptosis, and glial activation. Pretreatment with Botulinum Toxin A (BoNT/A) suppresses the Nox2–Hv1 axis, reduces ROS and inflammatory signaling, preserves retinal ganglion cells (Brn3a), and attenuates gliosis (Iba1, GFAP). Moreover, BoNT/A enhances protective mediators (SOCS3, GAP43, Syntaxin12), thereby shifting the retinal microenvironment from a degenerative toward a neuroprotective state.

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Modulation of the Nox2–Hv1–ROS Axis by Botulinum Neurotoxin A Confers Protection Against CoCl 2 -Induced Retinal Hypoxic Injury

doi: 10.3390/ijms262110806

Figure Lengend Snippet: Suppression of the Nox2–Hv1 Axis and Enhancement of Neuroprotective Signaling by BoNT/A in hypoxic injury. Cobalt chloride (CoCl 2 )–induced hypoxia stabilizes HIF-1α and triggers excessive ROS production, leading to metabolic stress within cells. This condition further drives inflammation (Hv1, Nox2, NLRP3, COX2, TNF-α), retinal thinning, apoptosis, and glial activation. Pretreatment with Botulinum Toxin A (BoNT/A) suppresses the Nox2–Hv1 axis, reduces ROS and inflammatory signaling, preserves retinal ganglion cells (Brn3a), and attenuates gliosis (Iba1, GFAP). Moreover, BoNT/A enhances protective mediators (SOCS3, GAP43, Syntaxin12), thereby shifting the retinal microenvironment from a degenerative toward a neuroprotective state.

Article Snippet: Syntaxin12 , Rabbit , Proteintech, Rosemont, IL, USA , 14259-1-AP , 1:200 (ICC), 1:1000 (WB).

Techniques: Activation Assay

E T9I has increased affinity to autophagosome-associated proteins (A) Principal component analysis of the differential interactome data , the individual replicates are separated (black: GFP controls, Green: E T9 pulldown, Purple: E T9I pulldown) (B) Volcano plot of the differential interactome analysis showing enriched proteins in E T9I pulldown versus the p value (-log P). Five highly significantly enriched proteins are highlighted in red and via labels. (C–G) Quantification of proximity ligation assays between transiently expressed SARS-CoV-2 E variants 30 h post transfection in HeLa cells and endogenous SNX12, STX12, TMEM87B, ABCG2 and TAB1, as indicated. Representative images depicted. PLA signal, red. Scale Bar, 10μm. DAPI, nuclei (blue). Lines represent the mean of N = 18–59 (individual cells) ±SEM. (H) Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEK293T-GL) transiently expressing StrepII-tagged SARS-CoV-2 E variants (48 h post transfection) and depleted of indicated proteins by siRNA. Bars represent the mean of N = 3 (biological replicates) ±SEM. Student’s t test with Welch’s correction. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Journal: iScience

Article Title: Mutation T9I in Envelope confers autophagy resistance to SARS-CoV-2 Omicron

doi: 10.1016/j.isci.2025.112974

Figure Lengend Snippet: E T9I has increased affinity to autophagosome-associated proteins (A) Principal component analysis of the differential interactome data , the individual replicates are separated (black: GFP controls, Green: E T9 pulldown, Purple: E T9I pulldown) (B) Volcano plot of the differential interactome analysis showing enriched proteins in E T9I pulldown versus the p value (-log P). Five highly significantly enriched proteins are highlighted in red and via labels. (C–G) Quantification of proximity ligation assays between transiently expressed SARS-CoV-2 E variants 30 h post transfection in HeLa cells and endogenous SNX12, STX12, TMEM87B, ABCG2 and TAB1, as indicated. Representative images depicted. PLA signal, red. Scale Bar, 10μm. DAPI, nuclei (blue). Lines represent the mean of N = 18–59 (individual cells) ±SEM. (H) Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEK293T-GL) transiently expressing StrepII-tagged SARS-CoV-2 E variants (48 h post transfection) and depleted of indicated proteins by siRNA. Bars represent the mean of N = 3 (biological replicates) ±SEM. Student’s t test with Welch’s correction. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Polyclonal rabbit anti-STX12 Antibody (PLA 1:100) (WB 1:500) (IP) , Proteintech , Cat#14259-1-AP; RRID: AB_2198222.

Techniques: Ligation, Transfection, Flow Cytometry, Expressing

Fig. 1 STX12 deficiency induces mitochondrial membrane potential defects in zebrafish. A A time-course plot of percent survival in the control vs. Stx12 morphants for 3 days. dpf, days postfertilization; hpf, hours postfertilization. B Representative images of five selected stages of zebrafish embryos. Scale bar: 100 μm. C qRT-PCR for five embryo development stages (0.2 hpf, 1 hpf, 2 hpf, 3.7 hpf and 6 hpf) demonstrating different expression patterns of Stx12 during embryonic development. D Representative images of TMRM staining and AO staining in control, E4I4-MO and ATG-MO zebrafish. Treatment window: 2 hpf-3.7 hpf; Stage of image: 3.7 hpf. TMRM staining: 1 μM; AO staining: 5 μg/mL. n = 10, Scale bar: 100 μm. E Quantification of the relative TMRM fluorescence intensity. F Quantification of apoptosis particle number in AO staining of control, E4I4-MO and ATG-MO zebrafish. Image analysis was performed using ImageJ software, with n = 10 per group. G Representative images of TMRM staining and AO staining after CCCP treatment. Stage of Image: 3.7hpf. Scale bar: 300 μm. Data are presented as mean ± SEM; statistical significance was assessed by Student’s t-test

Journal: Cell communication and signaling : CCS

Article Title: Pulmonary mitochondrial DNA release and activation of the cGAS-STING pathway in Lethal Stx12 knockout mice.

doi: 10.1186/s12964-025-02141-y

Figure Lengend Snippet: Fig. 1 STX12 deficiency induces mitochondrial membrane potential defects in zebrafish. A A time-course plot of percent survival in the control vs. Stx12 morphants for 3 days. dpf, days postfertilization; hpf, hours postfertilization. B Representative images of five selected stages of zebrafish embryos. Scale bar: 100 μm. C qRT-PCR for five embryo development stages (0.2 hpf, 1 hpf, 2 hpf, 3.7 hpf and 6 hpf) demonstrating different expression patterns of Stx12 during embryonic development. D Representative images of TMRM staining and AO staining in control, E4I4-MO and ATG-MO zebrafish. Treatment window: 2 hpf-3.7 hpf; Stage of image: 3.7 hpf. TMRM staining: 1 μM; AO staining: 5 μg/mL. n = 10, Scale bar: 100 μm. E Quantification of the relative TMRM fluorescence intensity. F Quantification of apoptosis particle number in AO staining of control, E4I4-MO and ATG-MO zebrafish. Image analysis was performed using ImageJ software, with n = 10 per group. G Representative images of TMRM staining and AO staining after CCCP treatment. Stage of Image: 3.7hpf. Scale bar: 300 μm. Data are presented as mean ± SEM; statistical significance was assessed by Student’s t-test

Article Snippet: The primary antibodies used included the following: rabbit anti-STX12 monoclonal antibody (1:1000, custom-made), mouse anti-β-actin (1:5000; Proteintech, #60,008–1-Ig), mouse anti-GAPDH (1:20,000, Proteintech, #60,004–1-Ig), anti-OXPHOS antibody (1:1000; Abcam, #ab110413, Cambridge, UK), anti-cGAS (1:1000; Cell Signaling Technology, #31,659, Danvers, MA, USA), anti-TBK1(1:1000, Cell Signaling Technology, #3504S), anti-pTBK1(1:1000; Cell Signaling Technology, # 5483S), anti-IRF3(1:1000; Proteintech, #11,312–1-AP), antipIRF3(1:1000; Cell Signaling Technology, #29,047), antiIL-6 (1:1000; Abcam, #ab229381), anti-S100A9 (1:1000; Boster, #PB0718, Wuhan, China).

Techniques: Membrane, Control, Quantitative RT-PCR, Expressing, Staining, Fluorescence, Software

Fig. 6 Schematic of systemic immune-inflammation in STX12-KO mice. The ablation of STX12 leads to decreased mitochondrial membrane potential (MMP), reduced expression levels of mitochondrial complex subunits, and the release of mitochondrial DNA (mtDNA). Then, mtDNA release activates the cGAS-STING-pTBK1-pIRF3 pathway, subsequently triggering Type I interferon response and downstream interferon-stimulated genes (ISGs) and cytokines in lung tissue. Additionally, cytokines release and neutrophil infiltration mutually enhance each other, resulting in an amplified cascade of hyperinflammation, referred to as “cytokine storm”, which potentially contributes to the mortality observed in Stx12 knockout mice

Journal: Cell communication and signaling : CCS

Article Title: Pulmonary mitochondrial DNA release and activation of the cGAS-STING pathway in Lethal Stx12 knockout mice.

doi: 10.1186/s12964-025-02141-y

Figure Lengend Snippet: Fig. 6 Schematic of systemic immune-inflammation in STX12-KO mice. The ablation of STX12 leads to decreased mitochondrial membrane potential (MMP), reduced expression levels of mitochondrial complex subunits, and the release of mitochondrial DNA (mtDNA). Then, mtDNA release activates the cGAS-STING-pTBK1-pIRF3 pathway, subsequently triggering Type I interferon response and downstream interferon-stimulated genes (ISGs) and cytokines in lung tissue. Additionally, cytokines release and neutrophil infiltration mutually enhance each other, resulting in an amplified cascade of hyperinflammation, referred to as “cytokine storm”, which potentially contributes to the mortality observed in Stx12 knockout mice

Techniques: Membrane, Expressing, Amplification, Knock-Out

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