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TaKaRa plvx dsred monomer n1 vector
Plvx Dsred Monomer N1 Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vector+plvx+dsred+monomer+n1/pLVX-DsRed-Monomer-N1+Vector/bio_rxiv__64898__2026__01__20__700440-239-8-10
Average 94 stars, based on 46 article reviews

plvx dsred monomer n1 vector - by Bioz Stars, 2026-09

94/100 stars

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Plasmid Preparation:

Article Title: Antitumor virus
Article Snippet: .. The plasmid encoding full-length KRM1 was constructed with reference to the similar method of Example 4, that is, the amino acids 1-473 coding sequence (Genbank number: AAH63787) was ligated into the vector pLVX-DsRed-Monomer-N1 (Clontech) through 5′Xhol and 3′NotI restriction sites, and a Flag tag and stop codon was added at the 3′ end. ..

Construct:

Sequencing:

FLAG-tag:

Image Search Results

(A) Kaplan-Meier analysis showing that neutrophil density negatively correlates with patient outcome, n=83 patients. (B) CD66b + TAN were analyzed by immunofluorescence and quantified in tumor, tumor margin and stroma of HNSCC patients (mean ± SD, n=83, circles = males, open circles = females). (C) Schematic overview of the in vitro system for tumor-stroma communication, generated using BioRender. (D) SNs from tumor (FaDu, PCI13), MSC-primed tumor, MSCs and tumor-primed MSCs were analysed for released CXCL8 by ELISA, mean + SD, n=3. (E) The impact of these SN on PMN chemotaxis was analyzed using transwell assays, mean + SD, n=3-8. (F) Luminex screening was performed on SNs from FaDu cells and seven patient-derived MSC lines, analyzed both in their naïve state and after FaDu-priming. CXCL8 was measured using ELISA. Data are depicted in pg/mL. Statistical analysis was performed with Kruskal-Wallis (B) and ordinary one-way ANOVA (D, E). P -values are indicated.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A) Kaplan-Meier analysis showing that neutrophil density negatively correlates with patient outcome, n=83 patients. (B) CD66b + TAN were analyzed by immunofluorescence and quantified in tumor, tumor margin and stroma of HNSCC patients (mean ± SD, n=83, circles = males, open circles = females). (C) Schematic overview of the in vitro system for tumor-stroma communication, generated using BioRender. (D) SNs from tumor (FaDu, PCI13), MSC-primed tumor, MSCs and tumor-primed MSCs were analysed for released CXCL8 by ELISA, mean + SD, n=3. (E) The impact of these SN on PMN chemotaxis was analyzed using transwell assays, mean + SD, n=3-8. (F) Luminex screening was performed on SNs from FaDu cells and seven patient-derived MSC lines, analyzed both in their naïve state and after FaDu-priming. CXCL8 was measured using ELISA. Data are depicted in pg/mL. Statistical analysis was performed with Kruskal-Wallis (B) and ordinary one-way ANOVA (D, E). P -values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Immunofluorescence, In Vitro, Generated, Enzyme-linked Immunosorbent Assay, Chemotaxis Assay, Luminex, Derivative Assay

(A) MSCs were stimulated for 24 h with FaDu tumor-conditioned SNs. Tumor-derived cytokines (columns) were quantified by Luminex, and MSC-derived CXCL8 and G-CSF (rows) by ELISA. Data are displayed as a correlation matrix. (B) MSCs were treated with FaDu tumor SNs for 24 h. Tumor-derived factors were analyzed by Luminex; MSC-derived CXCL8 and G-CSF were quantified by ELISA. Tumor-derived IL-1α correlates with MSC-derived CXCL8 and G-CSF. (C) MSCs were treated with recombinant IL-1α for 24 h. Release of CXCL8 and G-CSF was analyzed by ELISA. (D) IL-1α was measured in control (non-sense, NS) and IL-1α overexpressing FaDu cells (IL-1α-OE) using Luminex. (E) MSCs were treated with SNs from non-sense and IL-1α-OE cells. MSC-derived CXCL8 and G-CSF were quantified by ELISA. (F) IL-1α release was determined in the SN of viable and necrotic FaDu and A431 cells. MSCs were treated with the SN of viable and necrotic FaDu (G) or A431 (H) cells for 24 h. MSC-derived CXCL8 and G-CSF were quantified by ELISA. Statistical significance was assessed after log-transformation using an ordinary one-way ANOVA with Tukey’s multiple comparisons test (C), while paired t -tests were applied for panels D-H. Data are shown as mean ± SD. In panel C, significance levels are indicated as # or * (p ≤ 0.05), ## or ** (p ≤ 0.01), ### or *** (p ≤ 0.001), and #### or **** (p ≤ 0.0001); all other p values are shown numerically. Symbols (BioRender) are included to show the origin of the analyzed SNs.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A) MSCs were stimulated for 24 h with FaDu tumor-conditioned SNs. Tumor-derived cytokines (columns) were quantified by Luminex, and MSC-derived CXCL8 and G-CSF (rows) by ELISA. Data are displayed as a correlation matrix. (B) MSCs were treated with FaDu tumor SNs for 24 h. Tumor-derived factors were analyzed by Luminex; MSC-derived CXCL8 and G-CSF were quantified by ELISA. Tumor-derived IL-1α correlates with MSC-derived CXCL8 and G-CSF. (C) MSCs were treated with recombinant IL-1α for 24 h. Release of CXCL8 and G-CSF was analyzed by ELISA. (D) IL-1α was measured in control (non-sense, NS) and IL-1α overexpressing FaDu cells (IL-1α-OE) using Luminex. (E) MSCs were treated with SNs from non-sense and IL-1α-OE cells. MSC-derived CXCL8 and G-CSF were quantified by ELISA. (F) IL-1α release was determined in the SN of viable and necrotic FaDu and A431 cells. MSCs were treated with the SN of viable and necrotic FaDu (G) or A431 (H) cells for 24 h. MSC-derived CXCL8 and G-CSF were quantified by ELISA. Statistical significance was assessed after log-transformation using an ordinary one-way ANOVA with Tukey’s multiple comparisons test (C), while paired t -tests were applied for panels D-H. Data are shown as mean ± SD. In panel C, significance levels are indicated as # or * (p ≤ 0.05), ## or ** (p ≤ 0.01), ### or *** (p ≤ 0.001), and #### or **** (p ≤ 0.0001); all other p values are shown numerically. Symbols (BioRender) are included to show the origin of the analyzed SNs.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Derivative Assay, Luminex, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Transformation Assay

(A,B) Tumor-derived cytokines were quantified by Luminex analysis. (A) Data are displayed as Pearsońs correlation matrix. Significant correlations among tumor-derived factors are indicated by black asterisks (B). Quantification of tumor-derived mediators known to be involved in tumor-stroma communication. (C) MSCs were treated with FaDu-CXCL8KO tumor conditioned medium in the presence of 10 µg/mL IL-1α neutralizing antibody or isotype control. Released CXCL8 and G-CSF were quantified by ELISA. (D) Quantification of tumor-derived mediators of tumor-stroma communication in non-sense and IL-1α overexpressing (IL-1α-OE) cells was performed by Luminex. (E) Quantification of tumor-derived factors implicated in neutrophil recruitment and survival in SN from non-sense and IL-1α-OE cells (Luminex and CXCL8-ELISA). (F) LDH assay quantifying cytotoxicity in SN of viable and necrotic FaDu and A431 tumor cells. Statistical analysis was performed using paired t -tests (C,F) and unpaired t -tests (D). Data are presented as mean ± SEM (C) and mean ± SD (D,E), p values are indicated.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A,B) Tumor-derived cytokines were quantified by Luminex analysis. (A) Data are displayed as Pearsońs correlation matrix. Significant correlations among tumor-derived factors are indicated by black asterisks (B). Quantification of tumor-derived mediators known to be involved in tumor-stroma communication. (C) MSCs were treated with FaDu-CXCL8KO tumor conditioned medium in the presence of 10 µg/mL IL-1α neutralizing antibody or isotype control. Released CXCL8 and G-CSF were quantified by ELISA. (D) Quantification of tumor-derived mediators of tumor-stroma communication in non-sense and IL-1α overexpressing (IL-1α-OE) cells was performed by Luminex. (E) Quantification of tumor-derived factors implicated in neutrophil recruitment and survival in SN from non-sense and IL-1α-OE cells (Luminex and CXCL8-ELISA). (F) LDH assay quantifying cytotoxicity in SN of viable and necrotic FaDu and A431 tumor cells. Statistical analysis was performed using paired t -tests (C,F) and unpaired t -tests (D). Data are presented as mean ± SEM (C) and mean ± SD (D,E), p values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Derivative Assay, Luminex, Control, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

MSCs were treated with recombinant IL-1α for 6 and 24 h. Gene expression was analyzed by Bulk RNAseq. (A) Heatmap depicts functional DEGs linked to the biology and function of CAFs. (B) Enriched hallmark pathways are shown for untreated (ctrl) and 24 h IL-1α-treated MSCs. C) Pathway analysis (STRING v12.0) highlights local network cluster: IL-1 receptor activity and IL-1 family (yellow), chemokine receptors bind chemokines (green), extracellular matrix remodeling (pink), JAK-STAT (blue) and NFκB (red) signaling pathways. (D) Release of cytokines, chemokines and growth factors was analyzed by Luminex. Heatmap depicts proteins functioning in PMN survival, activation and chemotaxis. (E) Surface markers were analyzed by flow cytometry in different MSCs. (F) Inhibition of the Jak/Stat pathway by AZD1480 (JAK2i) or NFκB by Activation Inhibitor III (NFκBi) inhibit the release of CXCL8, G-CSF and IL6 in different MSCs. Red dots in E and F indicate the MSC cell line that was used for A-D. Statistical analysis was performed with paired t-tests. P -values are depicted.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: MSCs were treated with recombinant IL-1α for 6 and 24 h. Gene expression was analyzed by Bulk RNAseq. (A) Heatmap depicts functional DEGs linked to the biology and function of CAFs. (B) Enriched hallmark pathways are shown for untreated (ctrl) and 24 h IL-1α-treated MSCs. C) Pathway analysis (STRING v12.0) highlights local network cluster: IL-1 receptor activity and IL-1 family (yellow), chemokine receptors bind chemokines (green), extracellular matrix remodeling (pink), JAK-STAT (blue) and NFκB (red) signaling pathways. (D) Release of cytokines, chemokines and growth factors was analyzed by Luminex. Heatmap depicts proteins functioning in PMN survival, activation and chemotaxis. (E) Surface markers were analyzed by flow cytometry in different MSCs. (F) Inhibition of the Jak/Stat pathway by AZD1480 (JAK2i) or NFκB by Activation Inhibitor III (NFκBi) inhibit the release of CXCL8, G-CSF and IL6 in different MSCs. Red dots in E and F indicate the MSC cell line that was used for A-D. Statistical analysis was performed with paired t-tests. P -values are depicted.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Recombinant, Gene Expression, Functional Assay, Activity Assay, Protein-Protein interactions, Luminex, Activation Assay, Chemotaxis Assay, Flow Cytometry, Inhibition

MSCs were treated with recombinant IL-1α for 6 and 24 h. Gene expression was analyzed by Bulk RNAseq. Volcano plots are shown for untreated vs. 6 h (A), and untreated vs. 24 h IL-1α treatment (B). (C) Enriched hallmark pathways are shown for untreated vs. 6 h. (D) Venn diagram showing the number of upregulated DEGs in two MSC cell lines after 6 h of IL-1α treatment. (E) MSC cells were treated for 6 h and 24 h with IL-1α and were analyzed by RNAscope for the expression of CXCL8 (red) and CSF3 (green) with consecutive COX2 immunostaining (white). (F) FaDu spheroids were cultured in the presence or absence of SNs derived from MSCs or IL-1α-stimulated MSCs. Relative tumor spheroid area was quantified for two independent experiments. Statistical analysis was performed using Mann-Whitney test. Data depict mean ± SD. P -values are indicated.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: MSCs were treated with recombinant IL-1α for 6 and 24 h. Gene expression was analyzed by Bulk RNAseq. Volcano plots are shown for untreated vs. 6 h (A), and untreated vs. 24 h IL-1α treatment (B). (C) Enriched hallmark pathways are shown for untreated vs. 6 h. (D) Venn diagram showing the number of upregulated DEGs in two MSC cell lines after 6 h of IL-1α treatment. (E) MSC cells were treated for 6 h and 24 h with IL-1α and were analyzed by RNAscope for the expression of CXCL8 (red) and CSF3 (green) with consecutive COX2 immunostaining (white). (F) FaDu spheroids were cultured in the presence or absence of SNs derived from MSCs or IL-1α-stimulated MSCs. Relative tumor spheroid area was quantified for two independent experiments. Statistical analysis was performed using Mann-Whitney test. Data depict mean ± SD. P -values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Recombinant, Gene Expression, RNAscope, Expressing, Immunostaining, Cell Culture, Derivative Assay, MANN-WHITNEY

GSEA was performed on genes differentially expressed in MSCs after 24 h IL-1α treatment versus untreated controls. (A) Enrichment analysis of the IL-1α signature across eight CAF clusters . Clusters 3, 4, and 6 are associated with ECM remodeling, EMT, antigen presentation, and worse overall survival. (B) Enrichment analysis across five CAF clusters . HNCAF-0 (iCAF), HNCAF-3 (iCAF-like/ECM remodeling), and HNCAF-1 (iCAF, immunosuppressive) show high similarity to the IL-1α program; HNCAF-1 is linked to poor prognosis, whereas HNCAF-0/3 have been associated with favorable responses to anti-PD-1 immunotherapy. (C) The heatmap shows log2 fold changes of published CAF cluster marker genes in IL-1α-treated MSCs relative to control. The CF1/CXCL8 cluster represents an iCAF cluster and the most progressed fibroblast state.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: GSEA was performed on genes differentially expressed in MSCs after 24 h IL-1α treatment versus untreated controls. (A) Enrichment analysis of the IL-1α signature across eight CAF clusters . Clusters 3, 4, and 6 are associated with ECM remodeling, EMT, antigen presentation, and worse overall survival. (B) Enrichment analysis across five CAF clusters . HNCAF-0 (iCAF), HNCAF-3 (iCAF-like/ECM remodeling), and HNCAF-1 (iCAF, immunosuppressive) show high similarity to the IL-1α program; HNCAF-1 is linked to poor prognosis, whereas HNCAF-0/3 have been associated with favorable responses to anti-PD-1 immunotherapy. (C) The heatmap shows log2 fold changes of published CAF cluster marker genes in IL-1α-treated MSCs relative to control. The CF1/CXCL8 cluster represents an iCAF cluster and the most progressed fibroblast state.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Immunopeptidomics, Marker, Control

PMNs were treated with recombinant IL-1α and SNs from non-sense (NS) or IL-1α-overexpressing (OE) FaDu cells, as well as from MSCs preconditioned with these tumor cell SNs. A) PMN survival was analyzed by Annexin V/7-AAD staining (dotted line indicates the mean of untreated PMNs and (B) in the presence of neutralizing antibodies against G-CSF, GM-CSF, or both. (C,D) Activation was assessed using flow cytometry, determining changes in cell size (C) and granularity (D). (E) CCL4 release was quantified by ELISA. (F) Surface activation marker CD11b and CD54 were analyzed by flow cytometry. (G) ROS productionwas measured using 123-DiRhodamine. (H) Expression of marker genes was assessed by qRT-PCR. Statistical analysis was performed with paired t-tests (A,B), ordinary one-way ANOVA with Tukey’s multiple comparisons test (C) or Wilcoxon signed rank test (E-H). Data are plotted as mean + SD (E, G, H) or mean ± SD (A,B,F). P -values are stated.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: PMNs were treated with recombinant IL-1α and SNs from non-sense (NS) or IL-1α-overexpressing (OE) FaDu cells, as well as from MSCs preconditioned with these tumor cell SNs. A) PMN survival was analyzed by Annexin V/7-AAD staining (dotted line indicates the mean of untreated PMNs and (B) in the presence of neutralizing antibodies against G-CSF, GM-CSF, or both. (C,D) Activation was assessed using flow cytometry, determining changes in cell size (C) and granularity (D). (E) CCL4 release was quantified by ELISA. (F) Surface activation marker CD11b and CD54 were analyzed by flow cytometry. (G) ROS productionwas measured using 123-DiRhodamine. (H) Expression of marker genes was assessed by qRT-PCR. Statistical analysis was performed with paired t-tests (A,B), ordinary one-way ANOVA with Tukey’s multiple comparisons test (C) or Wilcoxon signed rank test (E-H). Data are plotted as mean + SD (E, G, H) or mean ± SD (A,B,F). P -values are stated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Recombinant, Staining, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Marker, Expressing, Quantitative RT-PCR

PMNs were treated with SN from non-sense (NS) or IL-1α-overexpressing (OE) FaDu cells, as well as from MSCs preconditioned with these tumor cell SN. (A) PMN migration toward tumor– and MSC-derived factors was analyzed by trans-well assays (dotted line indicates the mean of untreated PMNs) and (B) in the presence of neutralizing antibodies against CXCL8. (C) PMN migration was assessed by transwell assays toward SN collected from IL-1α–treated or untreated wild-type (WT) and CXCL8-knockout (KO) MSCs. (D) These SN were analyzed for released chemoattractants using Luminex. (E) Representative images of PMN recruitment into mixed NS FaDu-MSC or IL-1α OE-FaDu-MSC spheroids (tumor spheroids, blue; MSC spheroids, orange; PMNs, gray). (F) Quantification of PMN recruitment into tumor spheroids (NS or IL-1α-OE FaDu only), MSC spheroids (untreated vs. IL-1α-treated), and mixed tumor-MSC spheroids was analyzed by confocal microscopy (n = 6–10). (G) Representative images of FaDu cells (blue), MSCs (orange) and neutrophils (gray) 24 h post-injection into the perivitelline space of zebrafish larvae. Upper row: FaDu NS control cells; lower row: IL-1α-OE FaDu cells. Left panel: FaDu cells only; middle panel: FaDu cells + MSC1; right panel: FaDu cells + MSC2. (H) Quantification of infiltrated neutrophils per graft at 24 h post-injection. Statistical analysis was performed using paired t -tests (A-C), Mann–Whitney tests (F), or Wilcoxon signed-rank tests (H). Data are presented as mean ± SD (A-C), median (F), and mean ± SEM from three independent experiments (H). P values are indicated.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: PMNs were treated with SN from non-sense (NS) or IL-1α-overexpressing (OE) FaDu cells, as well as from MSCs preconditioned with these tumor cell SN. (A) PMN migration toward tumor– and MSC-derived factors was analyzed by trans-well assays (dotted line indicates the mean of untreated PMNs) and (B) in the presence of neutralizing antibodies against CXCL8. (C) PMN migration was assessed by transwell assays toward SN collected from IL-1α–treated or untreated wild-type (WT) and CXCL8-knockout (KO) MSCs. (D) These SN were analyzed for released chemoattractants using Luminex. (E) Representative images of PMN recruitment into mixed NS FaDu-MSC or IL-1α OE-FaDu-MSC spheroids (tumor spheroids, blue; MSC spheroids, orange; PMNs, gray). (F) Quantification of PMN recruitment into tumor spheroids (NS or IL-1α-OE FaDu only), MSC spheroids (untreated vs. IL-1α-treated), and mixed tumor-MSC spheroids was analyzed by confocal microscopy (n = 6–10). (G) Representative images of FaDu cells (blue), MSCs (orange) and neutrophils (gray) 24 h post-injection into the perivitelline space of zebrafish larvae. Upper row: FaDu NS control cells; lower row: IL-1α-OE FaDu cells. Left panel: FaDu cells only; middle panel: FaDu cells + MSC1; right panel: FaDu cells + MSC2. (H) Quantification of infiltrated neutrophils per graft at 24 h post-injection. Statistical analysis was performed using paired t -tests (A-C), Mann–Whitney tests (F), or Wilcoxon signed-rank tests (H). Data are presented as mean ± SD (A-C), median (F), and mean ± SEM from three independent experiments (H). P values are indicated.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: Migration, Derivative Assay, Knock-Out, Luminex, Confocal Microscopy, Injection, Control, MANN-WHITNEY

(A) Representative tumor section illustrating tumor-stroma segmentation, RNAscope detection of IL1A (pink), CXCL8 (red), and CSF3 (yellow), and zonal analysis of tumor-stroma communication. Shown is also an example of an IL1A + niche and an IL1A − niche with corresponding RNA probe signals. (B,C) Correlation of tumor IL1A expression with stromal CXCL8 + CSF3 + double-positive cells across the total cohort (B; n = 21; black circles = males, open circles = females) and in male patients only (C; n = 16). (D,E) Frequency of CXCL8 + CSF3 + double-positive stromal cells stratified by IL1A -negative vs. IL1A-positive tumors (D) and by TSR-low vs. TSR-high tumors (E). (F) Total TAN density and frequency in patients with low vs. high stromal CXCL8 + CSF3 + cells. (G) Spearman correlation analysis of an IL-1α-induced MSC/iCAF gene signature (SIG) with published TAN signatures across TCGA HNSCC tumor samples (left, n = 515) and matched tumor-adjacent normal tissue (right, n = 44). The MSC/iCAF signature (MSC-SIG) was defined by the top 100 genes upregulated in MSCs following 24 h IL-1α stimulation. TAN signatures from four independent studies – represent immature, inflammatory, and activated neutrophil populations. Individual cytokine and chemokine expression ( IL1A, IL1B, CSF2, CSF3, CXCL8 ) shows concordant associations with multiple TAN subset signatures. Statistical analysis was performed using Spearman correlation (B,C,G) and Mann–Whitney test (D-F). Data are presented scatter plots (B,C), or mean ± SEM (E–J). P values are indicated (A-D). In (G) p values are indicated as follow: * p value < 0.05, ** p value < 0.01, *** p value <0.001.

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet: (A) Representative tumor section illustrating tumor-stroma segmentation, RNAscope detection of IL1A (pink), CXCL8 (red), and CSF3 (yellow), and zonal analysis of tumor-stroma communication. Shown is also an example of an IL1A + niche and an IL1A − niche with corresponding RNA probe signals. (B,C) Correlation of tumor IL1A expression with stromal CXCL8 + CSF3 + double-positive cells across the total cohort (B; n = 21; black circles = males, open circles = females) and in male patients only (C; n = 16). (D,E) Frequency of CXCL8 + CSF3 + double-positive stromal cells stratified by IL1A -negative vs. IL1A-positive tumors (D) and by TSR-low vs. TSR-high tumors (E). (F) Total TAN density and frequency in patients with low vs. high stromal CXCL8 + CSF3 + cells. (G) Spearman correlation analysis of an IL-1α-induced MSC/iCAF gene signature (SIG) with published TAN signatures across TCGA HNSCC tumor samples (left, n = 515) and matched tumor-adjacent normal tissue (right, n = 44). The MSC/iCAF signature (MSC-SIG) was defined by the top 100 genes upregulated in MSCs following 24 h IL-1α stimulation. TAN signatures from four independent studies – represent immature, inflammatory, and activated neutrophil populations. Individual cytokine and chemokine expression ( IL1A, IL1B, CSF2, CSF3, CXCL8 ) shows concordant associations with multiple TAN subset signatures. Statistical analysis was performed using Spearman correlation (B,C,G) and Mann–Whitney test (D-F). Data are presented scatter plots (B,C), or mean ± SEM (E–J). P values are indicated (A-D). In (G) p values are indicated as follow: * p value < 0.05, ** p value < 0.01, *** p value <0.001.

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques: RNAscope, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

doi: 10.64898/2026.01.20.700440

Figure Lengend Snippet:

Article Snippet: For imaging immortalized MSCs were lentiviral transduced with pLVX-DsRed-Monomer-N1 vector (Takara Bio Inc., Saint-Germain-en-Laye, France) and selected via puromycin.

Techniques:

VMP1 is post–translationally modified by ubiquitination. ( a ) HeLa cells expressing empty pEGFPN1 or pEGFPN1 VMP1, or non–transfected HeLa cells were immunolabeled with anti–ubiquitin. Scale bars: 10 μm. The number of ubiquitin (Ub) dots per cell was quantified in at least 12 individual cells per condition in three independent experiments. The means per condition and the SEM are represented in the graphic. *** p < 0.001 according to a Kruskall–Wallis test. ( b ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti–ubiquitin. Scale bar: 10 μm. Colocalization between VMP1 and ubiquitin was quantified in 10 individual cells in three independent experiments. In the graphic, the red plots show Pearson’s correlation coefficient (PCC). *** p < 0.001 according to a two–tailed Student’s t –test vs. the theoretical mean, 0.5. ( c ) Lysates from HEK293T cells transfected with FLAG–Ub, VMP1–GFP, and the combination of both were immunoprecipitated with anti–FLAG magnetic beads and immunoblotted with anti–VMP1. Several bands over VMP1–GFP’s molecular weight (73 kDa) appeared in the eluate of co–transfected cells, indicating VMP1 ubiquitination. The image is representative of three independent experiments. ( d ) Lysates from HEK293T cells transfected with VMP1–GFP were immunoprecipitated with anti–GFP magnetic beads and immunoblotted with anti–Ub. Several bands over VMP1–GFP’s molecular weight (73 kDa) appeared in the eluate, indicating VMP1 ubiquitination.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: VMP1 is post–translationally modified by ubiquitination. ( a ) HeLa cells expressing empty pEGFPN1 or pEGFPN1 VMP1, or non–transfected HeLa cells were immunolabeled with anti–ubiquitin. Scale bars: 10 μm. The number of ubiquitin (Ub) dots per cell was quantified in at least 12 individual cells per condition in three independent experiments. The means per condition and the SEM are represented in the graphic. *** p < 0.001 according to a Kruskall–Wallis test. ( b ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti–ubiquitin. Scale bar: 10 μm. Colocalization between VMP1 and ubiquitin was quantified in 10 individual cells in three independent experiments. In the graphic, the red plots show Pearson’s correlation coefficient (PCC). *** p < 0.001 according to a two–tailed Student’s t –test vs. the theoretical mean, 0.5. ( c ) Lysates from HEK293T cells transfected with FLAG–Ub, VMP1–GFP, and the combination of both were immunoprecipitated with anti–FLAG magnetic beads and immunoblotted with anti–VMP1. Several bands over VMP1–GFP’s molecular weight (73 kDa) appeared in the eluate of co–transfected cells, indicating VMP1 ubiquitination. The image is representative of three independent experiments. ( d ) Lysates from HEK293T cells transfected with VMP1–GFP were immunoprecipitated with anti–GFP magnetic beads and immunoblotted with anti–Ub. Several bands over VMP1–GFP’s molecular weight (73 kDa) appeared in the eluate, indicating VMP1 ubiquitination.

Article Snippet: The human VMP1 sequence (NM_030938.5) was cloned into pENTR1F–flag2 using the GatewayTM LR ClonaseTM II (cat. No. 11791-020). pLVX-DsRed-Monomer-N1–hVMP1 was obtained by inserting the human VMP1 sequence (NCBI reference sequence: NM_030938.5) into pLVX-DsRed-Monomer-N1 (CLONTECH cat. 632152). pLenti–VMP1–GFP was obtained by cloning the VMP1 sequence (NCBI reference sequence: NM_030938.5) into pENTR1A–GFP-N2 (FR1) (Addgene Plasmid #19364).

Techniques: Modification, Expressing, Transfection, Immunolabeling, Two Tailed Test, Immunoprecipitation, Magnetic Beads, Molecular Weight

VMP1 is not monoubiquitinated with Myc–Ub (K0). ( a ) Schematic representation of Myc–Ub (K0) and Myc–Ub (wt) showing that Myc–Ub (K0) had its seven lysine residues replaced by arginine residues. In this way, Myc–Ub (K0) is only able to achieve monoubiquitination. ( b ) Lysates from HEK293T cells transfected with VMP1–GFP and Myc–Ub (wt) or Myc–Ub (K0) were immunoprecipitated with anti–Myc linked to protein G–Sepharose. The immunoblot with anti–GFP showed that there were no ubiquitination bands in the eluates from Myc–Ub (K0)–expressing cells, indicating that VMP1 is not monoubiquitinated. In contrast, eluates from Myc–Ub (wt)–expressing cells showed several bands corresponding to the polyubiquitination of VMP1. The image is representative of three independent experiments. K0 Ub: lysine–free ubiquitin; wt: wild type.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: VMP1 is not monoubiquitinated with Myc–Ub (K0). ( a ) Schematic representation of Myc–Ub (K0) and Myc–Ub (wt) showing that Myc–Ub (K0) had its seven lysine residues replaced by arginine residues. In this way, Myc–Ub (K0) is only able to achieve monoubiquitination. ( b ) Lysates from HEK293T cells transfected with VMP1–GFP and Myc–Ub (wt) or Myc–Ub (K0) were immunoprecipitated with anti–Myc linked to protein G–Sepharose. The immunoblot with anti–GFP showed that there were no ubiquitination bands in the eluates from Myc–Ub (K0)–expressing cells, indicating that VMP1 is not monoubiquitinated. In contrast, eluates from Myc–Ub (wt)–expressing cells showed several bands corresponding to the polyubiquitination of VMP1. The image is representative of three independent experiments. K0 Ub: lysine–free ubiquitin; wt: wild type.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

VMP1 is not degraded by the ubiquitin–proteasomal system, nor by autophagy. ( a ) Western blotting for VMP1, ubiquitin, and actin, where actin served as a loading control. HEK293T cells were transfected with VMP1–GFP and treated with or without the proteasome inhibitor MG132 (10 μM) over 6 h. The images are representative of three independent experiments. ( b ) Quantification of relative densitometry shows that VMP1–GFP (VMP1–GFP/actin) does not change significantly after treatment with MG132. ( c ) Relative densitometry (ubiquitin/actin) shows that ubiquitin levels increase significantly after MG132 treatment, indicating that the proteasome was effectively blocked. ** p < 0.01 vs. basal according to a Mann–Whitney nonparametric test; n = 3. ( d ) Western blotting for GFP and α–tubulin, where α–tubulin served as a loading control. HEK293T cells were transfected with VMP1–GFP and treated with 100 μg/mL of cycloheximide (CHX) over 0, 2, 4, 8, or 16 h. The images are representative of three independent experiments. ( e ) The graphic shows the means and SEMs of relative densitometry (VMP1–GFP/α–tubulin) in relation to the relative densitometry at 0 h; n = 3 for each experiment. ( f ) Western blotting for GFP and α–tubulin. HEK293T cells were transfected with VMP1–GFP and treated with CHX (100 μg/mL) and MG132 (10 μM) over 0, 2, 4, 8, or 16 h. The images are representative of three independent experiments. ( g ) The graphic shows the means and SEMs of relative densitometry (VMP1–GFP/α–tubulin) in relation to the relative densitometry at 0 h; n = 3 for each experiment. ( h ) Western blotting for VMP1, microtubule-associated protein 1 light–chain 3 (LC3), and actin, where actin served as a loading control. HEK 293T cells were transfected with VMP1–GFP and treated with or without 50 μM of the lysosome inhibitor chloroquine (CQ) over 24 h. The images are representative of three independent experiments. ( i ) Quantification of relative densitometry showed that VMP1–GFP (VMP1–GFP/actin) does not accumulate after treatment with CQ. In fact, VMP1–GFP significantly decreased in CQ–treated cells. * p < 0.05 vs. basal according to a Mann–Whitney nonparametric test; n = 3. ( j ) Relative densitometry (LC3–II/actin) showed that LC3–II levels significantly increased after CQ treatment, indicating that the lysosomal pathway was effectively blocked. *** p < 0.001 vs. basal according to a Mann–Whitney nonparametric test; n = 3. ( k ) Western blotting for GFP, LC3, and α–tubulin. HEK293T cells were transfected with VMP1–GFP and treated with CHX (100 μg/mL) and CQ (100 μM) over 0, 2, 4, or 8 h, or CHX 100 (μg/mL) and CQ (50 μM) over 16 h. The images are representative of three independent experiments. ( l ) The graphic shows the means and SEMs of relative densitometry (VMP1–GFP/α–tubulin) in relation to the relative densitometry at 0 h; n = 3 for each experiment.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: VMP1 is not degraded by the ubiquitin–proteasomal system, nor by autophagy. ( a ) Western blotting for VMP1, ubiquitin, and actin, where actin served as a loading control. HEK293T cells were transfected with VMP1–GFP and treated with or without the proteasome inhibitor MG132 (10 μM) over 6 h. The images are representative of three independent experiments. ( b ) Quantification of relative densitometry shows that VMP1–GFP (VMP1–GFP/actin) does not change significantly after treatment with MG132. ( c ) Relative densitometry (ubiquitin/actin) shows that ubiquitin levels increase significantly after MG132 treatment, indicating that the proteasome was effectively blocked. ** p < 0.01 vs. basal according to a Mann–Whitney nonparametric test; n = 3. ( d ) Western blotting for GFP and α–tubulin, where α–tubulin served as a loading control. HEK293T cells were transfected with VMP1–GFP and treated with 100 μg/mL of cycloheximide (CHX) over 0, 2, 4, 8, or 16 h. The images are representative of three independent experiments. ( e ) The graphic shows the means and SEMs of relative densitometry (VMP1–GFP/α–tubulin) in relation to the relative densitometry at 0 h; n = 3 for each experiment. ( f ) Western blotting for GFP and α–tubulin. HEK293T cells were transfected with VMP1–GFP and treated with CHX (100 μg/mL) and MG132 (10 μM) over 0, 2, 4, 8, or 16 h. The images are representative of three independent experiments. ( g ) The graphic shows the means and SEMs of relative densitometry (VMP1–GFP/α–tubulin) in relation to the relative densitometry at 0 h; n = 3 for each experiment. ( h ) Western blotting for VMP1, microtubule-associated protein 1 light–chain 3 (LC3), and actin, where actin served as a loading control. HEK 293T cells were transfected with VMP1–GFP and treated with or without 50 μM of the lysosome inhibitor chloroquine (CQ) over 24 h. The images are representative of three independent experiments. ( i ) Quantification of relative densitometry showed that VMP1–GFP (VMP1–GFP/actin) does not accumulate after treatment with CQ. In fact, VMP1–GFP significantly decreased in CQ–treated cells. * p < 0.05 vs. basal according to a Mann–Whitney nonparametric test; n = 3. ( j ) Relative densitometry (LC3–II/actin) showed that LC3–II levels significantly increased after CQ treatment, indicating that the lysosomal pathway was effectively blocked. *** p < 0.001 vs. basal according to a Mann–Whitney nonparametric test; n = 3. ( k ) Western blotting for GFP, LC3, and α–tubulin. HEK293T cells were transfected with VMP1–GFP and treated with CHX (100 μg/mL) and CQ (100 μM) over 0, 2, 4, or 8 h, or CHX 100 (μg/mL) and CQ (50 μM) over 16 h. The images are representative of three independent experiments. ( l ) The graphic shows the means and SEMs of relative densitometry (VMP1–GFP/α–tubulin) in relation to the relative densitometry at 0 h; n = 3 for each experiment.

Techniques: Western Blot, Transfection, MANN-WHITNEY

VMP1 associated with ubiquitin is involved in the autophagic process. ( a ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti-ubiquitin and anti–LC3. Scale bar: 10 μm. The images are representative of three independent experiments. The area bounded by the dotted line shows a perinuclear zone where the VMP1 that was not associated with Ub did not recruit LC3. ( b ) The square magnifications show examples of VMP1 dots that are associated with Ub and LC3. ( c ) Quantification of LC3 dots per cell associated with ubiquitin, VMP1, or ubiquitin and VMP1. *** p < 0.001 according to two–way ANOVA vs. Ub and VMP1 groups. n = 10 cells in three independent experiments. ( d ) Lysates from HEK293T cells that transfected with VMP1–GFP, FLAG–Ub and RFP–LC3 were immunoprecipitated with anti–GFP magnetic beads. The eluates were immunoblotted with anti–FLAG and anti–LC3. LC3 immunoblot shows a band of 41 kDa indicating the presence of RFP–LC3–II in the eluates. FLAG immunoblot shows the presence of ubiquitinated VMP1–GFP. ( e ) HeLa cells were transfected with VMP1–DsRed and GFP–DFCP1 and immunolabeled with anti–Ub. Scale bar: 10 μm. Arrows point to VMP1 dots that are associated with Ub and DFCP1. ( f ) Quantification of VMP1 dots per cell associated with DFCP1, ubiquitin, or ubiquitin and DFCP1. The graphic shows the means and SEMs. n = 10 cells in three independent experiments.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: VMP1 associated with ubiquitin is involved in the autophagic process. ( a ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti-ubiquitin and anti–LC3. Scale bar: 10 μm. The images are representative of three independent experiments. The area bounded by the dotted line shows a perinuclear zone where the VMP1 that was not associated with Ub did not recruit LC3. ( b ) The square magnifications show examples of VMP1 dots that are associated with Ub and LC3. ( c ) Quantification of LC3 dots per cell associated with ubiquitin, VMP1, or ubiquitin and VMP1. *** p < 0.001 according to two–way ANOVA vs. Ub and VMP1 groups. n = 10 cells in three independent experiments. ( d ) Lysates from HEK293T cells that transfected with VMP1–GFP, FLAG–Ub and RFP–LC3 were immunoprecipitated with anti–GFP magnetic beads. The eluates were immunoblotted with anti–FLAG and anti–LC3. LC3 immunoblot shows a band of 41 kDa indicating the presence of RFP–LC3–II in the eluates. FLAG immunoblot shows the presence of ubiquitinated VMP1–GFP. ( e ) HeLa cells were transfected with VMP1–DsRed and GFP–DFCP1 and immunolabeled with anti–Ub. Scale bar: 10 μm. Arrows point to VMP1 dots that are associated with Ub and DFCP1. ( f ) Quantification of VMP1 dots per cell associated with DFCP1, ubiquitin, or ubiquitin and DFCP1. The graphic shows the means and SEMs. n = 10 cells in three independent experiments.

Techniques: Transfection, Immunolabeling, Immunoprecipitation, Magnetic Beads, Western Blot

VMP1 remains ubiquitinated in the autophagic flux until it reaches the autolysosome. ( a ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti-LAMP2. Scale bars: 10 μm. ( b ) Colocalization between VMP1 and lysosome–associated membrane glycoprotein 2 (LAMP2) was quantified by Pearson’s correlation coefficient (PCC). Similar results were obtained in three independent experiments. The red plots show PCC for individual cells; n = 10. *** p < 0.001 according to a Kruskall–Wallis test. ( c ) HeLa cells were transfected with VMP1–DsRed and LAMP1–YFPs and immunolabeled with anti–LC3. Scale bar: 10 μm. Arrows point to VMP1 dots that are associated with lysosome–associated membrane glycoprotein 1 (LAMP1) and LC3. ( d ) Quantification of VMP1 dots per cell associated with LC3, LAMP1, and LC3–LAMP1. The graphic shows the means and SEMs. n = 10 cells in three independent experiments. ( e ) HeLa cells were transfected with VMP1–DsRed and GFP–DFCP1 and labeled with Lysotracker Blue DND–22. Scale bar: 10 μm. ( f ) The graphic shows the means and SEMs of the VMP1 dots associated with DFCP1, Lysotracker, or DFCP1 and Lysotracker. n = 10 cells in three independent experiments. ( g ) HeLa cells were transfected with VMP1–DsRed and LAMP1–YFPs and immunolabeled with anti–Ub. Scale bar: 10 μm. Arrows point to VMP1 dots that are associated with Ub and LAMP1. ( h ) Quantification of VMP1 dots per cell associated with ubiquitin, LAMP1, and Ub–LAMP1. The graphic shows the means and SEMs. n = 10 cells in three independent experiments. YFP: yellow fluorescent protein.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: VMP1 remains ubiquitinated in the autophagic flux until it reaches the autolysosome. ( a ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti-LAMP2. Scale bars: 10 μm. ( b ) Colocalization between VMP1 and lysosome–associated membrane glycoprotein 2 (LAMP2) was quantified by Pearson’s correlation coefficient (PCC). Similar results were obtained in three independent experiments. The red plots show PCC for individual cells; n = 10. *** p < 0.001 according to a Kruskall–Wallis test. ( c ) HeLa cells were transfected with VMP1–DsRed and LAMP1–YFPs and immunolabeled with anti–LC3. Scale bar: 10 μm. Arrows point to VMP1 dots that are associated with lysosome–associated membrane glycoprotein 1 (LAMP1) and LC3. ( d ) Quantification of VMP1 dots per cell associated with LC3, LAMP1, and LC3–LAMP1. The graphic shows the means and SEMs. n = 10 cells in three independent experiments. ( e ) HeLa cells were transfected with VMP1–DsRed and GFP–DFCP1 and labeled with Lysotracker Blue DND–22. Scale bar: 10 μm. ( f ) The graphic shows the means and SEMs of the VMP1 dots associated with DFCP1, Lysotracker, or DFCP1 and Lysotracker. n = 10 cells in three independent experiments. ( g ) HeLa cells were transfected with VMP1–DsRed and LAMP1–YFPs and immunolabeled with anti–Ub. Scale bar: 10 μm. Arrows point to VMP1 dots that are associated with Ub and LAMP1. ( h ) Quantification of VMP1 dots per cell associated with ubiquitin, LAMP1, and Ub–LAMP1. The graphic shows the means and SEMs. n = 10 cells in three independent experiments. YFP: yellow fluorescent protein.

Techniques: Transfection, Immunolabeling, Labeling

$Cdt2 is involved in the ubiquitination of VMP1. ( a ) Schematic representation of the E3 ligase complex cullin–RING ubiquitin ligase complex 4 (CRL4)/Cdt2, in which the cell division cycle protein cdt2 (Cdt2) works as the substrate recognition subunit. ( b ) Table of main interactors of Cdt2 identified by MS of the sequential immunoprecipitation with anti–FLAG and anti–HA of soluble-fraction (non–chromatin–fraction) HEK293T cells transfected with FLAG–HA–Cdt2. VMP1 was found between the interactors. ( c ) Lysates from HEK293T cells transfected with Myc–Cdt2 and VMP1–FLAG were immunoprecipitated with anti–FLAG magnetic beads and immunoblotted with anti–Myc. ( d ) Lysates from HEK293T cells transfected with FLAG–Cdt2 were immunoprecipitated with anti–FLAG magnetic beads and immunoblotted with anti–VMP1. ( e ) HeLa cells were immunolabeled with anti–Cdt2. Scale bar: 10 μm. ( f ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti-Cdt2. Scale bar: 10 μm. Arrowheads point to VMP1 dots that co–distribute with Cdt2. ( g ) Endogenous Cdt2 localization was measured as the ratio between the extranuclear and intranuclear integrated density of fluorescence in VMP1–GFP– and non–transfected HeLa cells. *** p < 0.001 vs. control according to a Mann–Whitney nonparametric test. ( h ) Lysates from HEK293T cells transfected with VMP1–GFP and Myc–Ub with or without Cdt2–FLAG, and treated or not with MLN4924 (1 μM, 5 h), were immunoprecipitated with anti–Myc linked to protein G–Sepharose. The eluates were immunoblotted with anti–GFP. Ubiquitinated VMP1 increased in the presence of Cdt2–FLAG and decreased under treatment with MLN4924. Myc–Ub (K0) was used as a negative control. The images are representative of three independent experiments.$

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: Cdt2 is involved in the ubiquitination of VMP1. ( a ) Schematic representation of the E3 ligase complex cullin–RING ubiquitin ligase complex 4 (CRL4)/Cdt2, in which the cell division cycle protein cdt2 (Cdt2) works as the substrate recognition subunit. ( b ) Table of main interactors of Cdt2 identified by MS of the sequential immunoprecipitation with anti–FLAG and anti–HA of soluble-fraction (non–chromatin–fraction) HEK293T cells transfected with FLAG–HA–Cdt2. VMP1 was found between the interactors. ( c ) Lysates from HEK293T cells transfected with Myc–Cdt2 and VMP1–FLAG were immunoprecipitated with anti–FLAG magnetic beads and immunoblotted with anti–Myc. ( d ) Lysates from HEK293T cells transfected with FLAG–Cdt2 were immunoprecipitated with anti–FLAG magnetic beads and immunoblotted with anti–VMP1. ( e ) HeLa cells were immunolabeled with anti–Cdt2. Scale bar: 10 μm. ( f ) HeLa cells were transfected with VMP1–GFP and immunolabeled with anti-Cdt2. Scale bar: 10 μm. Arrowheads point to VMP1 dots that co–distribute with Cdt2. ( g ) Endogenous Cdt2 localization was measured as the ratio between the extranuclear and intranuclear integrated density of fluorescence in VMP1–GFP– and non–transfected HeLa cells. *** p < 0.001 vs. control according to a Mann–Whitney nonparametric test. ( h ) Lysates from HEK293T cells transfected with VMP1–GFP and Myc–Ub with or without Cdt2–FLAG, and treated or not with MLN4924 (1 μM, 5 h), were immunoprecipitated with anti–Myc linked to protein G–Sepharose. The eluates were immunoblotted with anti–GFP. Ubiquitinated VMP1 increased in the presence of Cdt2–FLAG and decreased under treatment with MLN4924. Myc–Ub (K0) was used as a negative control. The images are representative of three independent experiments.

Techniques: Immunoprecipitation, Transfection, Magnetic Beads, Immunolabeling, Fluorescence, MANN-WHITNEY, Negative Control

The distribution of VMP1 and its ability to trigger autophagy are both affected by treatment with MLN4924, a CRL inhibitor. ( a ) HeLa cells were treated with MLN4924 (1 μM, 5 h) or solvent. The cells were then immunolabeled with anti–LC3. Representative images are shown. Scale bar: 10 μm. ( b ) The number of LC3 dots per cell was quantified in both the control and MLN4924–treated cells. N = 13 cells per condition from three independent experiments. ( c ) Western blotting for LC3 and α–tubulin where α–tubulin served as a loading control. HEK293T cells were transfected with Empty–GFP and either treated with MLN4924 (1 μM, 5 h) or solvent. Images are representative of three independent experiments. ( d ) Quantification of relative densitometry shows LC3–II (LC3–II/α–tubulin) does not change significantly after treatment with MLN4924. N:3. ( e ) HeLa cells were transfected with pLenti–VMP1–GFP and either treated with MLN4924 (1 μM, 5 h) or solvent. The cells were then immunolabeled with anti–LC3. Representative images are shown. Scale bar: 10 μm. ( f ) The number of VMP1 dots per cell was significantly reduced in MLN4924–treated cells. The graph shows the means and SEMs of 18 control cells and 22 MLN4924–treated cells across three independent experiments. *** p < 0.001 vs. Control by Mann–Whitney test. ( g ) The number of LC3 dots per cell was also significantly reduced in MLN4924–treated cells compared to control. The means and SEMs are shown in the graph. The analysis was based on 18 control cells and 17 MLN4924–treated cells across three independent experiments. * p < 0.05 vs. Control by Mann–Whitney test. ( h ) Western blotting for LC3 and α–tubulin where α–tubulin served as a loading control. HEK293T cells were transfected with pLenti–VMP1–GFP and either treated with MLN4924 (1 μM, 5 h) or solvent. Images are representative of four independent experiments ( i ) Quantification of relative densitometry shows LC3–II (LC3–II/α–tubulin) significantly decrease after treatment with MLN4924. * p < 0.05 vs. Control by Student’s t test. N = 4.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: The distribution of VMP1 and its ability to trigger autophagy are both affected by treatment with MLN4924, a CRL inhibitor. ( a ) HeLa cells were treated with MLN4924 (1 μM, 5 h) or solvent. The cells were then immunolabeled with anti–LC3. Representative images are shown. Scale bar: 10 μm. ( b ) The number of LC3 dots per cell was quantified in both the control and MLN4924–treated cells. N = 13 cells per condition from three independent experiments. ( c ) Western blotting for LC3 and α–tubulin where α–tubulin served as a loading control. HEK293T cells were transfected with Empty–GFP and either treated with MLN4924 (1 μM, 5 h) or solvent. Images are representative of three independent experiments. ( d ) Quantification of relative densitometry shows LC3–II (LC3–II/α–tubulin) does not change significantly after treatment with MLN4924. N:3. ( e ) HeLa cells were transfected with pLenti–VMP1–GFP and either treated with MLN4924 (1 μM, 5 h) or solvent. The cells were then immunolabeled with anti–LC3. Representative images are shown. Scale bar: 10 μm. ( f ) The number of VMP1 dots per cell was significantly reduced in MLN4924–treated cells. The graph shows the means and SEMs of 18 control cells and 22 MLN4924–treated cells across three independent experiments. *** p < 0.001 vs. Control by Mann–Whitney test. ( g ) The number of LC3 dots per cell was also significantly reduced in MLN4924–treated cells compared to control. The means and SEMs are shown in the graph. The analysis was based on 18 control cells and 17 MLN4924–treated cells across three independent experiments. * p < 0.05 vs. Control by Mann–Whitney test. ( h ) Western blotting for LC3 and α–tubulin where α–tubulin served as a loading control. HEK293T cells were transfected with pLenti–VMP1–GFP and either treated with MLN4924 (1 μM, 5 h) or solvent. Images are representative of four independent experiments ( i ) Quantification of relative densitometry shows LC3–II (LC3–II/α–tubulin) significantly decrease after treatment with MLN4924. * p < 0.05 vs. Control by Student’s t test. N = 4.

Techniques: Immunolabeling, Western Blot, Transfection, MANN-WHITNEY

Schematic model. VMP1 is ubiquitinated in the initial steps of autophagosome biogenesis and remains ubiquitinated throughout the autophagic flux until autolysosome formation. Moreover, VMP1 interacts with Cdt2, the adaptor subunit of the CRL4/Cdt2 E3 ligase complex, which is able to catalyze the VMP1 ubiquitination. ER: endoplasmic reticulum.

Journal: International Journal of Molecular Sciences

Article Title: Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells

doi: 10.3390/ijms241612981

Figure Lengend Snippet: Schematic model. VMP1 is ubiquitinated in the initial steps of autophagosome biogenesis and remains ubiquitinated throughout the autophagic flux until autolysosome formation. Moreover, VMP1 interacts with Cdt2, the adaptor subunit of the CRL4/Cdt2 E3 ligase complex, which is able to catalyze the VMP1 ubiquitination. ER: endoplasmic reticulum.

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