Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments modulate Notch receptors expression and activity. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (A) TALL-1 or (C) MOLT3 cells treated for 48 h with 2 μM GSKJ4 or with DMSO. (B) Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panel) and HA and β-actin protein levels (lower panel) in TALL-1 cells transfected with HA-tagged EZH2 expression vector (HA-EZH2) or with the empty control vector. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (D) TALL-1 or (E) MOLT3 cells treated for 48 h with 5 μM A-485 or DMSO. Data represent mean values of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, ** P < 0.01, *** P < 0.001. Uncropped western blots related to this figure are displayed in .

Article Snippet: The expression vector PIRVNeoSV containing the human c-Myc cDNA coding sequence (c-Myc) was kindly provided by Dr. Giuseppe Giannini (Sapienza University, Rome, Italy). pCMV3-HA vector containing the human EZH2 coding sequence (HA-EZH2) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Nature Communications

Article Title: Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis

doi: 10.1038/s41467-025-62723-9

Figure Lengend Snippet: A Method that SDS-PAGE and Coomassie brilliant blue (CBB) staining of RIPK3-immunoprecipitated proteins from HEK 293T cells stably overexpressing FLAG-RIPK3. Icons of this figure created with BioRender.com. B Proteins that we identified by mass spectrometry (MS) analysis. Icons of this figure created with BioRender.com. C HT-29 cells were treated with 20 ng/mL TNFα, 2 μM Birinapant, and 20 μM Z-VAD-FMK (T/Bi/Z) for 2 h and 3 h. After treatment, the cell lysates were immunoprecipitated with RIPK3 antibody and analyzed using immunoblotting. D HT-29 cells were treated with T/Bi/Z for 2 h and 3 h. After treatment, the cell lysates were immunoprecipitated with SMURF1 antibody and analyzed using immunoblotting. E HT-29 cells were treated with T/Bi/Z for 2 h and 3 h. After treatment, the cell lysates were immunoprecipitated with SMURF1 antibody and analyzed using immunoblotting. F HT-29 cells were treated with T/Bi/Z for 3 h. The cell lysates were fractionated according to molecular size by gel-filtration chromatography and examined using immunoblotting. G HT-29 cells were treated with T/Bi/Z for 3 h. After treatment, the cells were fixed and stained using anti-SMURF1 and p-RPIPK3 antibodies, and DAPI. H HT-29 cells were treated with T/Bi/Z for 3 h. After treatment, the cells were fixed and stained using anti-USP5 and p-RIPK3 antibodies, and DAPI. Scale bars = 10 μm.

Article Snippet: The SMURF1 and USP5 plasmid was purchased from Sinobiological (Beijing, China, #HG13144-NY, MG51371-CF). pcs4-3xMYC-SMURF1 mutants (C699A), pcs4-3xMYC-USP5 mutants (C335A) were generated using site-directed mutagenesis and PCR, respectively.

Techniques: SDS Page, Staining, Immunoprecipitation, Stable Transfection, Mass Spectrometry, Western Blot, Filtration, Chromatography

Journal: Nature Communications

Article Title: Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis

doi: 10.1038/s41467-025-62723-9

Figure Lengend Snippet: A HT-29 cells were transduced with lentivirus to generate stable SMURF1-knockdown (KD) cell lines. The expression levels of RIPK1, RIPK3, MLKL, and SMURF1 in each cell line were analyzed by western blotting. B HT-29 cells depleted of SMURF1 using the indicated shRNAs were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK'963 for 2.5 h. Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the Annexin V-FITC positive region. This experiment was independently repeated 3 times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. C SMURF1 KD and control HT-29 cells were treated with T/Bi/Z to induce necroptosis. GSK'963 was used to confirm that this cell death is necroptosis. Cells were harvested at the indicated time points and analyzed by western blotting. D SMURF1 KD and control HT-29 cells treated with T/Bi/Z were lysed after 2 h. The necrosome was pulled down using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed and analyzed by western blotting to assess the assembly of the RIPK1-RIPK3-MLKL complex. The asterisk indicates p-MLKL. E shSMURF1-infected cells, reconstituted with either SMURF1 WT or the E3 ligase-inactive form (C699A), were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK'963 for 2 h. Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the Annexin V-FITC positive region. This experiment was independently repeated three times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. F shSMURF1-infected cells, reconstituted with either SMURF1 WT or the E3 ligase-inactive form (C699A), were treated with T/Bi/Z to induce necroptosis. GSK'963 was used to confirm that this cell death is necroptosis. Cells were harvested at the indicated time points and analyzed by western blotting. The asterisk indicates p-RIPK3. G shSMURF1-infected cells, reconstituted with either SMURF1 WT or the E3 ligase-inactive form (C699A), were treated with T/Bi/Z and lysed after 2 h. The necrosome was pulled down using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed and analyzed by western blotting to examine the assembly of the RIPK1-RIPK3-MLKL complex.

Article Snippet: The SMURF1 and USP5 plasmid was purchased from Sinobiological (Beijing, China, #HG13144-NY, MG51371-CF). pcs4-3xMYC-SMURF1 mutants (C699A), pcs4-3xMYC-USP5 mutants (C335A) were generated using site-directed mutagenesis and PCR, respectively.

Techniques: Transduction, Knockdown, Expressing, Western Blot, Flow Cytometry, Control, Infection

Journal: Nature Communications

Article Title: Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis

doi: 10.1038/s41467-025-62723-9

Figure Lengend Snippet: A FLAG-RIPK1, RIPK3, MLKL and 3xMYC-SMURF1 were overexpressed in HEK 293T cells via PEI transfection. Cells were lysed and incubated with anti-FLAG antibodies and protein-G agarose beads to pull down 3xMYC-SMURF1.The protein-bead conjugate was washed and analyzed by SDS-PAGE. The immunoprecipitation of the SMURF1 and RIPK1, RIPK3, MLKL were assessed using western blotting. B FLAG-RIPK3 was transfected into HEK 293T cells along with HA-Ub, with or without 3xMYC-SMURF1 expression. After 20 h, cells were lysed in a 1% SDS buffer, and lysates diluted to 0.1% SDS were immunoprecipitated using an anti-FLAG antibody. RIPK3 ubiquitylation was detected by western blotting with an HRP-conjugated anti-HA antibody. C FLAG-RIPK3 (1–296, kinase domain; 294–518, C-term domain; 1–440 and 452–518 ΔRHIM domian) and 3xMYC-SMURF1 were overexpressed in HEK 293T cells via PEI transfection. Cells were lysed and incubated with anti-FLAG antibodies and protein-G agarose beads to pull down 3xMYC-SMURF1. The protein-bead conjugate was washed and analyzed by SDS-PAGE. The immunoprecipitation of the SMURF1 and RIPK3 domains was assessed using western blotting. The schematic diagram of RIPK3 shows the binding domain for SMURF1. Icons of this figure created with BioRender.com. D HA-SMURF1 (1–159, 119–731, 1–345, 346–731, 119–365) and FLAG-RIPK3 were overexpressed in HEK 293 T cells via PEI transfection. Cells were lysed and incubated with anti-HA antibodies and protein-G agarose beads to pull down the specific domain of HA-SMURF1. The protein-bead conjugate was washed and analyzed by SDS-PAGE. The immunoprecipitation of the RIPK3 and SMURF1 domains was assessed using western blotting. The schematic diagram of RIPK3 shows the binding domain for RIPK3. Icons of this figure created with BioRender.com. E HEK 293 T cells were transfected with FLAG-RIPK3, 3xMYC–SMURF1, and HA-Ub along with various ubiquitin mutants. After 20 h, cells were lysed in a 1% SDS buffer, and lysates diluted to 0.1% SDS were immunoprecipitated using an anti-FLAG antibody. RIPK3 ubiquitylation was detected by western blotting with an HRP-conjugated anti-HA antibody. F HEK 293T cells were transfected with FLAG-RIPK3, 3xMYC–SMURF1, and HA-Ub along with the K63R ubiquitin mutant. After 20 h, cells were lysed in a 1% SDS buffer, and lysates diluted to 0.1% SDS were immunoprecipitated using an anti-FLAG antibody. RIPK3 ubiquitylation was detected by western blotting with an HRP-conjugated anti-HA antibody. G To confirm the endogenous ubiquitination status of RIPK3, HT-29 cells expressing shGFP or shSMURF1 (#4 and #8) were treated with T/Bi/Z to induce necroptosis. After 3 h, SMURF1 KD and control HT-29 cells were lysed. Immunoprecipitation was performed using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed, and ubiquitination was detected using an anti-Ub-HRP conjugated antibody and an anti-K63-Ub-HRP conjugated antibody.

Article Snippet: The SMURF1 and USP5 plasmid was purchased from Sinobiological (Beijing, China, #HG13144-NY, MG51371-CF). pcs4-3xMYC-SMURF1 mutants (C699A), pcs4-3xMYC-USP5 mutants (C335A) were generated using site-directed mutagenesis and PCR, respectively.

Techniques: Transfection, Incubation, SDS Page, Immunoprecipitation, Western Blot, Expressing, Binding Assay, Ubiquitin Proteomics, Mutagenesis, Control

Journal: Nature Communications

Article Title: Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis

doi: 10.1038/s41467-025-62723-9

Figure Lengend Snippet: A This is a schematic diagram illustrating the various ubiquitination enzyme sites on RIPK3 identified to date. Icons of this figure created with BioRender.com. B FLAG-RIPK3 WT, K55R, K197R, K302R, K363R, K364R, K518R, and K55/363R were transfected into HEK 293T cells with HA-Ub, with or without 3xMYC-SMURF1 expression. After 20 h, cells were lysed in a 1% SDS buffer, and lysates diluted to 0.1% SDS were immunoprecipitated using an anti-FLAG antibody. RIPK3 ubiquitination was detected by western blotting with an HRP-conjugated anti-HA antibody. C Adenovirus-mediated expression of RIPK3 WT, K55R, K363R, and K55/363R was induced in HeLa cells, which were then treated with T/Bi/Z to induce necroptosis. After 5 h, the cells were harvested and stained with propidium iodide (PI). Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC(Side Scatter), and calculating the percentage of cells in the PI(Propidium Iodide) positive region. This experiment was independently repeated 3 times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. D HeLa cells expressing RIPK3 WT, K55R, K363R, and K55/363R were treated with T/Bi/Z to induce necroptosis at the indicated time points. Cells were harvested at these time points and analyzed by western blotting. The asterisk indicates p-RIPK3. E HeLa cells expressing shGFP, shSMURF1, RIPK3 WT, and RIPK3 K55/363R were treated with T/Bi/Z to induce necroptosis. After 5 h, the cells were harvested and stained with propidium iodide (PI). Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the PI(Propidium Iodide) positive region. This experiment was independently repeated 3 times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. F HeLa cells expressing shGFP, shSMURF1, RIPK3 WT, and RIPK3 K55/363R were treated with T/Bi/Z to induce necroptosis. After 4 h, the cells were harvested and analyzed by western blotting.

Article Snippet: The SMURF1 and USP5 plasmid was purchased from Sinobiological (Beijing, China, #HG13144-NY, MG51371-CF). pcs4-3xMYC-SMURF1 mutants (C699A), pcs4-3xMYC-USP5 mutants (C335A) were generated using site-directed mutagenesis and PCR, respectively.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Immunoprecipitation, Western Blot, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis

doi: 10.1038/s41467-025-62723-9

Figure Lengend Snippet: A HT-29 cells depleted of SMURF1, USP5 and Double Knocked down by the indicated shRNAs. B HT-29 cells depleted of SMURF1, USP5 and Double Knocked down by the indicated shRNAs were treated with T/Bi/Z in the presence or absence of 0.1 μM GSK’963 for 3 h. The cells were harvested and stained with annexin V-FITC. Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the Annexin V-FITC positive region. This experiment was independently repeated 3 times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. C Each HT-29 cells depleted of SMURF1, USP5 and Double Knocked down cells were treated with T/Bi/Z to induce necroptosis. GSK’963 (0.1 μM) was used to inhibit necroptosis. Cells were harvested and analyzed using western blotting. D Each HT-29 cells depleted of SMURF1, USP5 and Double Knocked down cells treated with T/Bi/Z were lysed after 3 h. The necrosome was pulled down using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed and analyzed by western blotting to assess the assembly of the RIPK1-RIPK3-MLKL complex. E To confirm the endogenous ubiquitination status of RIPK3, HT-29 cells depleted of SMURF1, USP5 and Double Knocked down were treated with T/Bi/Z to induce necroptosis. After 3 h, SMURF1 KD and control HT-29 cells were lysed. Immunoprecipitation was performed using an anti-RIPK3 antibody and protein-G agarose beads. The protein-bead conjugate was washed, and ubiquitination was detected using an anti-Ub-HRP conjugated antibody and an anti-K63-Ub-HRP conjugated antibody.

Article Snippet: The SMURF1 and USP5 plasmid was purchased from Sinobiological (Beijing, China, #HG13144-NY, MG51371-CF). pcs4-3xMYC-SMURF1 mutants (C699A), pcs4-3xMYC-USP5 mutants (C335A) were generated using site-directed mutagenesis and PCR, respectively.

Techniques: Staining, Flow Cytometry, Western Blot, Ubiquitin Proteomics, Control, Immunoprecipitation

Journal: Nature Communications

Article Title: Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis

doi: 10.1038/s41467-025-62723-9

Figure Lengend Snippet: A Molm-13 cells were transduced with lentivirus to generate stable SMURF1-KD cell lines. The expression levels of RIPK1, RIPK3, MLKL, and SMURF1 in each cell line were analyzed by western blotting. B Molm-13 depleted of SMURF1 using the indicated shRNAs were treated with Bi/E in the presence or absence of 0.1 μM GSK'963 for 12 h. The cells were harvested and stained with PI. Flow cytometry was performed by counting 10,000 cells based on FSC (Forwarder Scatter) and SSC (Side Scatter), and calculating the percentage of cells in the PI (Propidium Iodide) positive region. This experiment was independently repeated 3 times. Data are presented as means ± standard deviations (SDs), n = 3. Significance between groups was determined using two-sided Students’ test. C SMURF1 KD and control Molm-13 cells were treated with Bi/E to induce necroptosis. GSK'963 was used to confirm that this cell death is necroptosis. Cells were harvested at the indicated time points and analyzed by western blotting. D Tumor xenografts were established by injecting control or SMURF1-knockdown (KD) Molm-13 cell lines into the flanks of mice (n = 6). This experimental setup aimed to examine the effect of SMURF1 depletion on tumor progression and response to treatment. E Following xenograft establishment, the mice received intraperitoneal injections of birinapant (2 mg/kg) and emricasan (1 mg/kg) every 4 days for a total of six treatments. Six independent mice were used for xenograft experiments in each experimental group, and treatments were administered independently for each group. Representative images of the resulting xenografts were taken to visually document tumor development. Tumor growth was systematically measured at 4-day intervals throughout the treatment period to monitor any differences between control and SMURF1 KD groups. Data are presented as means ± standard deviations (SDs). Significance was determined using the two-sided Students’ test. F At the end of the experiment, the tumors were excised, and their masses were recorded to evaluate the impact of SMURF1 depletion on tumor size. The tumor weight was measured from each of the six independent mice in each group. Data are presented as means ± standard deviations (SDs). Significance was determined using the two-sided Students’ test. G The tumors were lysed, and SMURF1 expression was analyzed using western blotting. H Additional tumor xenografts were generated using NB4 cell lines expressing either RIPK3 WT or the RIPK3 K55/363R mutant (n = 6). This setup aimed to assess the role of specific RIPK3 mutations in tumor growth and response to therapeutic agents. I Mice bearing these xenografts were treated with birinapant (2 mg/kg) and emricasan (1 mg/kg) via intraperitoneal injection every 5 days, for a total of eight treatments. Six independent mice were used for xenograft experiments in each experimental group, and treatments were administered independently for each group. Tumor growth was measured at 5-day intervals throughout the course of the experiment to track progression over time in both RIPK3 WT and K55/363R groups. Data are presented as means ± standard deviations (SDs). Significance was determined using the two-sided Students’ test. J Upon completion of the experiment, tumors were dissected, and their weights were measured to quantify differences between groups. The tumor weight was measured from each of the six independent mice in each group. Tumor weights were compared to determine any statistically significant variations attributable to SMURF1 KD or RIPK3 mutations.

Article Snippet: The SMURF1 and USP5 plasmid was purchased from Sinobiological (Beijing, China, #HG13144-NY, MG51371-CF). pcs4-3xMYC-SMURF1 mutants (C699A), pcs4-3xMYC-USP5 mutants (C335A) were generated using site-directed mutagenesis and PCR, respectively.

Techniques: Transduction, Expressing, Western Blot, Staining, Flow Cytometry, Control, Knockdown, Generated, Mutagenesis, Injection

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: AP2M1’s Role in TRPV1’s SUMOylation-Dependent Membrane Localization. (A) Exploration of the AP2M1 protein-protein interaction network using the STRING database. CoIP-MS analysis was performed on protein lysates from MGC-803 cells stably expressing Flag-hTRPV1 WT and Flag-hTRPV1 K823R , identifying differential binding partners in TRPV1 K823R versus TRPV1 WT cell lines. (B) SUMOylation contributed to AP2M1’s modulation of TRPV1 membrane expression in GC xenografts. Tissue lysates from GC xenografts of WT and K823R groups underwent Co-IP using Flag beads, followed by IB with anti-AP2M1 and anti-Flag antibodies (biological replicates ≥ 3). (C) In vivo SUMOylation’s involvement in AP2M1-mediated changes in TRPV1 membrane expression. Tissue lysates from spontaneous gastric tumorigenesis in WT and KI mice were analyzed through Co-IP with either control IgG or anti-AP2M1 antibody, followed by IB with anti-AP2M1 and anti-TRPV1 (biological replicates ≥ 3). (D) Disrupting TRPV1-AP2M1 interaction enhanced TRPV1’s membrane presence in MGC-803 cells. Membrane proteins from stably transfected MGC-803 cells were biotinylated, isolated with streptavidin-agarose, and assessed by IB on 12% SDS-polyacrylamide gels using anti-Flag and on 8% SDS-polyacrylamide gels using anti-TfR antibodies (biological replicates ≥ 3). (E) AP2M1 knockdown eliminated TRPV1-AP2M1 association in MGC-803 cells. Following co-transfection with AP2M1 siRNA and either Flag-hTRPV1 WT or Flag-hTRPV1 K823R , cell lysates were subjected to precipitation with Flag beads and analyzed by Western blot to detect protein interactions (biological replicates ≥ 3). (F) AP2M1 knockdown enhanced TRPV1’s membrane localization in MGC-803 cells. Membrane proteins from cells co-transfected with AP2M1 siRNA and either Flag-hTRPV1 WT or Flag-hTRPV1 K823R were isolated using the biotin-avidin method and quantified by IB using anti-Flag or anti-TfR following biotinylation and purification with streptavidin-agarose (biological replicates ≥ 3)

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Membrane, Stable Transfection, Expressing, Binding Assay, Co-Immunoprecipitation Assay, In Vivo, Control, Transfection, Isolation, Knockdown, Cotransfection, Western Blot, Avidin-Biotin Assay, Purification

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: Impact of Disrupting AP2M1-TRPV1 Interaction on GC Cell Migration and Proliferation. (A) Mapping the TRPV1 interaction domain on AP2M1. Four AP2M1 truncation mutants were generated: HA-AP2M1 1–261 , HA-AP2M1 262–435 , HA-AP2M1 1–165 , and HA-AP2M1 166–261 . MGC-803 cells were transiently co-transfected with Flag-hTRPV1 WT and these mutants along with the HA-AP2M1 plasmid. IP used an anti-Flag antibody, followed by IB with anti-HA and anti-Flag to detect interactions. Input levels were verified by IB using anti-Flag and anti-HA antibodies (biological replicates ≥ 3). (B) Depiction of HA-AP2M1 WT deletions, including segments Δ166–175, Δ176–185, Δ186–195, Δ196–205, Δ206–216, Δ217–226, Δ227–237, Δ238–248, and Δ249–261, to identify critical interaction regions. (C) The 176–185 region on AP2M1 was crucial for anchoring TRPV1. Co-transfection of MGC-803 cells with Flag-hTRPV1 WT and HA-AP2M1 WT or mutants, followed by Co-IP using Flag beads and subsequent HA probing, identified this segment as key for TRPV1 binding (biological replicates ≥ 3). (D) AP2M1’s interaction with TRPV1 enhanced TRPV1’s membrane localization via SUMOylation. Co-expression of Flag-hTRPV1 WT or Flag-hTRPV1 K823R with either HA-AP2M1 WT or HA-AP2M1 Δ176–185 in AP2M1 -deficient MGC-803 cells allowed for membrane TRPV1 level assessment through the biotin-avidin method. IB analysis followed, targeting biotinylated membrane proteins with anti-Flag or anti-TfR (biological replicates ≥ 3). (E) Interrupting the TRPV1-AP2M1 bond reduced MGC-803 cell migration as shown by transwell migration assays. Migration counts are presented as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, ns = no significant, analyzed by one-way ANOVA with Tukey’s test. Scale bar: 200 μm. (F) Colony formation assays revealed that disrupting TRPV1-AP2M1 interaction decreased the clonogenic potential of MGC-803 cells. Colony counts are summarized as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, ns = no significant, via one-way ANOVA with Tukey’s test

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Migration, Generated, Transfection, Plasmid Preparation, Cotransfection, Co-Immunoprecipitation Assay, Binding Assay, Membrane, Expressing, Avidin-Biotin Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: The TAT-AP2M1-176–185 Peptide Mitigates GC Pathogenicity by Modulating TRPV1 Localization and Function. (A) Design of TAT fusion peptides, including the hAP2M1 fragment 176–185 sequence and a scrambled sequence as a control, to investigate their effects on TRPV1-AP2M1 interactions. (B) Application of the TAT-AP2M1-176–185 peptide disrupted AP2M1’s binding to TRPV1 in MGC-803 cells expressing Flag-hTRPV1 WT or Flag-hTRPV1 K823R . Cells were treated with 20 µM of either TAT-AP2M1-176–185 or TAT-Scramble for 3 h, followed by Co-IP with an anti-Flag antibody and IB for AP2M1 detection (biological replicates ≥ 3). (C) Enhanced membrane expression of TRPV1 in MGC-803 cells post TAT-AP2M1-176–185 treatment, as measured by biotin-avidin purification and IB using anti-Flag or anti-TfR. Biological replicates ≥ 3. (D) Interruption of the TRPV1-AP2M1 interaction reduced MGC-803 cell migration, as shown by transwell migration assays. Data are presented as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, with no significant (ns) difference noted in certain comparisons, analyzed by one-way ANOVA with Tukey’s test. Scale bar: 200 μm. (E) Disruption of TRPV1-AP2M1 binding also decreased the colony-forming ability of MGC-803 cells. Results are summarized as mean ± SEM from ≥ 3 biological replicates; **** p < 0.0001, ns = no significant, one-way ANOVA with Tukey’s test. (F) Schematic of xenograft experiments where BALB/c nude mice were subcutaneously (S.C.) injected with MGC-803 cells and subsequently intraperitoneally injected with TAT-AP2M1-176–185 or TAT-Scramble after one week to assess effects on tumor growth and pathology. (G) Mice treated with TAT-AP2M1-176–185 exhibited slower tumor growth in GC xenografts compared to those receiving TAT-Scramble (biological replicates ≥ 3). (H) Quantification of tumor size revealed significant reductions in tumors treated with TAT-AP2M1-176–185 compared to TAT-Scramble, demonstrating the peptide’s efficacy in reducing tumor growth. Tumor dimensions were used to calculate volume (L × W^2)/2, with data presented as mean ± SEM for 9–10 mice; ** p < 0.01, **** p < 0.0001, analyzed by two-way ANOVA with Tukey’s test. (I) Tumor weights at the study endpoint were significantly lower in the TAT-AP2M1-176–185 treatment group than in the TAT-Scramble group, further evidencing the peptide’s impact. Data from (G) are summarized, with mean ± SEM for 9–10 mice; *** p < 0.001, ns = no significant, by one-way ANOVA with Tukey’s test. (J) TAT-AP2M1-176–185 effectively abolished AP2M1’s binding to TRPV1 in xenograft tumors, as shown by Co-IP and IB analyses post-treatment (biological replicates ≥ 3). (K) Enhanced membrane localization of TRPV1 in GC tumors treated with TAT-AP2M1-176–185 compared to TAT-Scramble, as determined by membrane protein extraction and IB (biological replicates ≥ 3)

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Sequencing, Control, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Membrane, Avidin-Biotin Assay, Purification, Migration, Disruption, Injection, Protein Extraction

Journal: Cell Communication and Signaling : CCS

Article Title: SUMOylation-induced membrane localization of TRPV1 suppresses proliferation and migration in gastric cancer cells

doi: 10.1186/s12964-024-01850-0

Figure Lengend Snippet: TRPV1 SUMOylation Suppresses GC Cell Proliferation and Migration by Activating the TRPV1-Ca 2+ -AMPK Pathway. (A) RNA sequencing (RNA-seq) performed on RNA extracted from MGC-803 cells stably expressing Flag-hTRPV1 WT and Flag-hTRPV1 K823R identified gene sets associated with the negative regulation of the AMPK signaling pathway. Gene set enrichment analysis (GSEA) indicated a differential expression favoring Flag-hTRPV1 K823R over Flag-hTRPV1 WT , with the normalized enrichment score (NES) and p-value shown. (B) Expression of phosphorylated AMPK (p-AMPK) relative to total AMPK in GC tissues from WT and K823R xenograft tumors was analyzed by Western blot. Quantification showed increased p-AMPK in WT compared to K823R (biological replicates ≥ 3); *** p < 0.001 by one-way ANOVA with Tukey’s test. (C) Normal stomach tissues from WT and KI mice were analyzed for p-AMPK/AMPK expression. No significant difference was observed; n ≥ 3 biological replications; ns = no significant difference by the two-tailed Student’s t -test. (D) GC tissues from spontaneous gastric tumorigenesis in WT and KI mice showed higher p-AMPK levels in WT; n ≥ 3 biological replications, ** p < 0.01 by the two-tailed Student’s t -test. (E) Disruption of TRPV1-AP2M1 interaction by deleting the N-terminal of TRPV1 (ΔN mutants) in MGC-803 cells resulted in increased AMPK activation; data present the mean ± SEM of n ≥ 3 biological replications; *** p < 0.001, **** p < 0.0001 by one-way ANOVA with Tukey’s test. (F) Similarly, co-transfection with HA-AP2M1 WT and HA-AP2M1 Δ176–185 demonstrated that disrupting TRPV1-AP2M1 binding enhanced AMPK activation; data present the mean ± SEM of n ≥ 3 biological replications; ** p < 0.01 by one-way ANOVA with Tukey’s test. (G) Treatment with TAT-AP2M1-176–185 peptide significantly increased AMPK activation in MGC-803 cells expressing TRPV1 variants, compared to the TAT-Scramble control; data present the mean ± SEM of n ≥ 3 biological replications; **** p < 0.0001 by one-way ANOVA with Tukey’s test. (H) In xenograft tumors from WT and K823R groups treated with TAT-AP2M1-176–185 or TAT-Scramble, p-AMPK levels were markedly higher in the TAT-AP2M1-176–185 treated group; data present the mean ± SEM of n ≥ 3 biological replications; **** p < 0.0001 by one-way ANOVA with Tukey’s test

Article Snippet: Additionally, we utilized the HA-AP2M1 plasmid (catalog number HG16144-NY) sourced from Sino Biological Inc., Shanghai, China.

Techniques: Migration, RNA Sequencing Assay, Stable Transfection, Expressing, Western Blot, Two Tailed Test, Disruption, Activation Assay, Cotransfection, Binding Assay, Control

Journal: Viruses

Article Title: Unveiling the Role of TMPRSS2 in the Proteolytic Activation of Pandemic and Zoonotic Influenza Viruses and Coronaviruses in Human Airway Cells

doi: 10.3390/v16111798

Figure Lengend Snippet: Activation of SARS-CoV and MERS-CoV S by TMPRSS2 and further host proteases. ( A , B ) HeLa cells were co-transfected with plasmids coding for MERS-CoV S ( A ) or SARS-CoV ( B ) with a C-terminal FLAG-tag and plasmids coding for human TMPRSS2 or HAT or murine TMPRSS4 or TMPRSS13. Cell lysates were analysed via SDS-PAGE at 48 h p.t. Western blot analysis was performed using an antibody targeting the C-terminal FLAG-tag. Tubulin was used as a loading control. The data shown are representative of three individual experiments. EV = empty vector. ( C , E ) HeLa cells co-expressing MERS-CoV ( C ) or SARS-CoV ( E ) S, as well as the respective receptors DPP4 or ACE2, were incubated for 48 h. After fixation, the cells were stained with a primary antibody targeting the C-terminal FLAG-tag of S and a fluorescence-coupled secondary antibody. DAPI was used to stain the nuclei. Representative images of three independent experiments are shown. The scale bar represents 100 µM. ( D , F ) Quantification of MERS-CoV S ( D ) or SARS-CoV ( F ) mediated cell–cell fusion. For each condition, ten randomly taken images were analysed by counting the nuclei per syncytium (with at least three nuclei). The data shown are the mean values + SEM of three independent experiments. Statistical significance was determined with a one-way ANOVA followed by Šídák’s multiple comparisons test. ns = not significant, ** = p < 0.01, **** = p < 0.0001.

Article Snippet: The expression vectors coding for SARS- or MERS-CoV S with a C-terminal FLAG-tag, pCMV3-MERS-Spike-cFLAG (VG40069-CF) and pCMV3-SARS-Spike-cFLAG (VG40150-CF), as well as a vector encoding human dipeptidyl peptidase 4 (DPP4) with a C-terminal HA-tag, pCMV3-hDPP4-cHA (HG10688-CY), were purchased from Sino Biological, Houston, TX, USA.

Techniques: Activation Assay, Transfection, FLAG-tag, SDS Page, Western Blot, Control, Plasmid Preparation, Expressing, Incubation, Staining, Fluorescence

Journal: bioRxiv

Article Title: The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-CoV-2 in cell culture models

doi: 10.1101/2021.01.10.426114

Figure Lengend Snippet: A. Schematic showing ACE2 tagged with LgBIT and SmBIT. B. HEK-293 cells were transfected with combinations of plasmids encoding LgBIT or SmBIT fused to either protein kinase A regulatory subunit (PRKAR2) or catalytic subunit (PRKACA), ATG5 or ACE2. The results (mean ± S.D., n = 5) were normalized to the luminescence measured in cells transfected with protein kinase A reporters (positive control). C. HEK-293 cells were transfected with plasmids encoding ACE2 nanoBIT reporters under the control of the HSV TK promoter and ACE2 or prolactin (PRL) under the control of the CMV promoter. The results (mean ± S.D., n = 4) were normalized to the luminescence measured in cells transfected with protein kinase A reporters and prolactin. D. HEK-293 cells were transfected with NanoBIT-tagged ACE2 reporters and incubated with sodium valproate or clofibrate at a concentration equal to 1x, 2x or 3x the reported C max of the drug. After 1 hour, luminescence was measured and normalized (mean ± S.D., n =4) to that measured in cells treated with DMSO. E. A series of other fibrates were similarly evaluated in the assay. The luminescence measured (mean ± S.D., n = 5-11, solid bars) was significantly different to that measured in cells treated with solvent where shown. When these fibrates were incubated with purified LgBIT and HiBIT-RBD to create a constitutively active nanoluc, each of these fibrates were found to inhibit nanoluciferase (bezafibrate 35 ± 7 %, ciprofibrate 55 ± 6 %, fenofibric acid 46 ± 3 %, fenofibrate 69 ± 5 %, gemfibrozil 61 ± 2 % of the activity measured in the presence of DMSO). To correct for this, the luminescence measurements from cells treated with fibrates in cells were divided by these latter values to estimate the effect of the drugs on dimerization (hatched bars). Significant difference from control is shown as *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Article Snippet: The plasmid pcDNA3 encoding ACE2 was obtained from GenScript (OHu20260); the plasmid encoding prolactin (PRL) was obtained from Sino Biological (HG10275-CY).

Techniques: Transfection, Positive Control, Incubation, Concentration Assay, Purification, Activity Assay