Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: Autophagy limits CAR‐T cell‐mediated cytotoxicity by suppressing TNF‐α induced apoptosis. (A) Volcano plot showing varied genes in Nalm6 cells with the addition of vehicle (as control) and autophinib (log 2 FC > 1 and P < 0.05). (B) Volcano plot showing varied genes between sgControl and indicated RB1CC1 KO Nalm6 cells (log 2 FC > 1 and P < 0.05). (C) KEGG pathway enrichment analysis of varied genes identified in RNA sequencing for Nalm6 cells treated with vehicle (as control) and autophinib. (D) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins after the addition of vehicle (as control), autophinib and SAR405 in Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (E) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins in sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1) Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (F) Expression of TNFRSF1A mRNA by RT‐qPCR and TNFR1 protein by western blotting in Nalm6 and Raji cells after addition of vehicle (as control), autophinib and SAR405 ( n = 3). Values are shown as the mean ± SD. Statistical differences among three groups in each cell line are calculated with one‐way ANOVA with tests. (G‐H) Cytotoxic analysis of sgControl and indicated gene‐KO (sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E:T ratio = 1:4) when treated with vehicle (as control), autophinib and SAR405 and then with or without TNF‐block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. (I) Cytotoxic analysis of sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1, sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E: T ratio = 1: 4) when treated with or without TNF block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T chimeric antigen receptor T; E:T effector:target; FC fold change; FC fold change; KO knockout; KEGG Kyoto Encyclopedia of Genes and Genomes; RT‐qPCR real‐time quantitative polymerase chain reaction; ns: not significant; SD standard deviation; sg single guide; TNF tumor necrosis factor; TNFR1 tumor necrosis factor receptor 1.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Control, RNA Sequencing, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Blocking Assay, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: STAT1/IRF1 axis mediates the upregulation of CXCL10 and CXCL11 induced by autophagy targeting. (A‐B) Western blotting showing the expression levels of STAT1, pSTAT1, and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors or the knockout of RB1CC1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (C‐D) Western blotting showing the expression levels of STAT1 and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors and the silencing of STAT1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (E‐F) Expression of CXCL10 and CXCL11 mRNA by RT‐qPCR and ELISA quantification of CXCL10 and CXCL11 protein levels in the supernatants of shControl, shSTAT1 and shIRF1 Nalm6 and Raji cells ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA tests. (G‐H) ChIP‐qPCR showing the binding of STAT1 and IRF1 to the promoter region of CXCL10 and CXCL11 ( n = 3). Values are shown as the mean ± SD. Statistical differences for each cell line are calculated with unpaired Student's t tests. (I) Graphic abstract: in the proposed model, inhibition of cancer cell‐autonomous autophagy leads to accumulation of cytosolic DNA, which thereby not only suppresses cancer cell survival by inducing TNFR1‐TNF‐α mediated apoptosis but also promotes the CAR‐T cell recruitment in tumor microenvironment via STAT1/IRF1‐dependent activation of chemokine signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T, chimeric antigen receptor T; ChIP, chromatin immunoprecipitation; CXCL CXC, chemokine ligand; ELISA, enzyme‐linked immunosorbent assay; FC, fold change; IRF, interferon regulatory factor; ns: not significant; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; sh short hairpin; STAT, signal transducers and activators of transcription.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Western Blot, Expressing, Knock-Out, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Binding Assay, Inhibition, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: bioRxiv

Article Title: Tumor necrosis factor receptor 2 inhibits HIV-1 infection by blocking the binding of gp120 to CD4

doi: 10.1101/2025.08.05.668613

Figure Lengend Snippet: TNFR1 impacts the binding of gp120 to CD4 but promotes the virus infection. (A) The binding interaction between gp120 and TNFR1 was assessed using an ELISA assay with varying concentrations of gp120 (5μM、0.5μM、0.05μM、 blank). (B) Flow cytometry was utilized to evaluate cell-bound His-tag, indicating the binding of gp120 to the Jurkat cells, and also assess if TNFR1 (0.4µM、0.6µM、 1µM) can block gp120-cell binding. (C) The percentage of positive cells determined from flow cytometry analysis results. (D) Flow cytometry was utilized to detect the binding of gp120 to human peripheral blood mononuclear cells and to evaluate the blocking effect of TNFR1. (E) The statistical analysis of the double positive cells. (F) HIV pseudovirus carrying green fluorescent protein genes were used to infect CD4+CCR5+293T cells (MOI=5、MOI=10). The flow cytometry was used to analyze the rate of GFP-positive cells to illustrate the virus infection efficiency. (G) The percentage of GFP positive cells determined from flow cytometry analysis results. (H) CD4⁻CCR5⁺ 293T, TNFR1 + CD4⁻CCR5⁺ 293T, and TNFR2 + CD4⁻CCR5⁺ 293T cells were infected with GFP-expressing HIV-1 pseudovirus. Infection efficiency was assessed by flow cytometry based on the percentage of GFP⁺ cells. (I) Quantification of infection efficiency. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns no significant difference.

Article Snippet: The human TNFR1 protein (DNA sequence: NP_001056 (Ile 30 - Thr 211) Catalog No: TN1-H5222) was purchased from the ACRO Biosystems (Beijing, China).

Techniques: Binding Assay, Virus, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Blocking Assay, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Protein-Bound Polysaccharides from Coriolus Versicolor Induce RIPK1/RIPK3/MLKL-Mediated Necroptosis in ER-Positive Breast Cancer and Amelanotic Melanoma Cells.

doi: 10.33594/000000242

Figure Lengend Snippet: Fig. 4. PBPs enhance autocrine production of TNF-α, which is required for the necroptosis of breast can- cer cells. (a) Viability and (b) ROS generation in MCF-7 cells pretreated (1 h) with neutralizing antibody (5 μg/ml) against TNF-α prior to treatment with PBPs (100 μg/ml or 200 μg/ml). (c) mRNA expression levels of TNF-α and TNFR1 in MCF-7 cells treated with PBPs for 24 h, as determined by quantitative real- time PCR. (d) Detection of TNFR1 expression (green) using immunohistochemical assays of MCF-7 cells incubated with PBS (control) or stimulated with PBPs (200 μg/ml) for 48 h. Cell nuclei were counterstained with PI (red).

Article Snippet: Primary rabbit polyclonal antibody against TNFR1 (cat. nr orb100329) was from Biorbyt Ltd. (UK), and goat anti-rabbit IgG (H+L) secondary antibody conjugated to Alexa Fluor 488 (cat. nr A-11034) was purchased from Thermo Fisher Scientific (USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Incubation, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Protein-Bound Polysaccharides from Coriolus Versicolor Induce RIPK1/RIPK3/MLKL-Mediated Necroptosis in ER-Positive Breast Cancer and Amelanotic Melanoma Cells.

doi: 10.33594/000000242

Figure Lengend Snippet: Fig. 5. PBPs do not enhance the autocrine production of TNF-α in amelanotic melanoma cells. (a) Viability and (b) ROS generation in SKMel-188 cells pretreated (1 h) with neutralizing antibody (5 μg/ml) against TNF-α prior to treatment with PBPs (100 μg/ml or 200 μg/ml). (c) The mRNA expression levels of TNF-α and TNFR1 in SKMel-188 cells treated with PBPs for 24 h, as determined by quantitative real-time PCR. (d) Detection of TNFR1 expression (green) using immunohistochemical assays with SKMel-188 cells in- cubated with PBS (control) or stimulated with PBPs (200 μg/ml) for 48 h. Cell nuclei were counterstained with PI (red).

Article Snippet: Primary rabbit polyclonal antibody against TNFR1 (cat. nr orb100329) was from Biorbyt Ltd. (UK), and goat anti-rabbit IgG (H+L) secondary antibody conjugated to Alexa Fluor 488 (cat. nr A-11034) was purchased from Thermo Fisher Scientific (USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Control

Journal: Cell Death & Disease

Article Title: A TNFR1–UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop

doi: 10.1038/s41419-022-05308-4

Figure Lengend Snippet: a Left: comparing the intensities of immunohistochemical (IHC) staining with an anti-TNFR1 antibody in 33 normal, 34 adjacent, and 32 lung SCC samples in an array (BC04118, Biomax). +++, strong staining; ++, moderate staining; +, weak staining. Comparison among these groups; **** P < 0.0001; Chi-squared test. Right: representative images of TNFR1 IHC staining in normal human lungs and lung SCCs. Numbers on the top of the images represent the location of the samples in the array (Fig. ). Scale bar: 50 μM. b TNFR1 protein levels detected by immunoblotting of human lung SCCs (SC) and in adjacent tissue samples (Ad). P, patient; β-actin, a protein-loading control. c TNFRSF1A expression levels, as detected by using RNA sequencing (RNA-seq), in a human lung SCC cohort (TCGA, PanCancer Atlas, n = 466, cBioPortal). d TNFRSF1A DNA alterations in three human lung SCC cohorts (1: TCGA, Firehose Legacy; 2: TCGA, Nature 2012; 3: TCGA, PanCancer Atlas, cBioPortal) and in three human lung ADC cohorts (1: TCGA, Firehose Legacy; 2: TCGA PanCancer Atlas; and 3: OncoSG, Nat Genet 2020, cBioPortal). Mean ± SEM (three repeats); Student’s t -test. e Co-expression of CHUK (IKKα) and TNFRSF1A in a human lung SCC cohort obtained from Cell 2021, CPTAC: Clinical Proteomic Tumor Consortium, (cBioPortal for Cancer Genomics). FPKM, Fragments Per Kilobase of transcript per Million mapped reads. P -values are indicated in this panel. f TNFR1 protein levels detected by immunoblotting in mouse lung SCCs isolated from KA/KA mice and in adjacent tissues. M, mouse; *, indicating a band that may represent an oligomer, a nonspecific band, or a cleaved TNFR1; β-actin, a protein-loading control. g Incidence of lung SCCs in KA/KA;Tnfrsf1a +/+ , KA/KA;Tnfrsf1a +/- , and KA/KA;Tnfrsf1a -/- mice. Numbers indicate mouse numbers. **** P < 0.0001; Chi square test. h Left: Generation of transgenic Tg-K5.TNFR1 mice. K5, keratin 5; Tg, transgenic. Right: Representative H&E-stained images of K5.TNFR1; KA/KA lung SCC and metastasis (Metast) to the liver, and of KA/KA lung SCC. Scale bar: 40 μM.

Article Snippet: A mouse TNFR1 full-length cDNA fragment (1,365 bp) in the CMV-Myc Flag TNFR1 vector (MR226545, OriGene, Rockville, MD) was purified after being digested with KpnI and PmeI .

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Comparison, Western Blot, Control, Expressing, RNA Sequencing, Isolation, Transgenic Assay

Journal: Cell Death & Disease

Article Title: A TNFR1–UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop

doi: 10.1038/s41419-022-05308-4

Figure Lengend Snippet: a Left: TNFR1 protein levels detected by immunoblotting in M2C, KAL LU- (Scal lo CD24 + CD44 + ) and KAL LU+ (Scal hi CD24 - CD44 + ) cells. lo, low; hi, high; β-actin, a protein-loading control. Right: Flow cytometry analyzes TNFR1 expression levels (MFI, Mean Fluorescence Intensity) in KAL LU- and KAL LU+ cells (high panel, images; low panel, statistical analysis for MFI of TNFR1 staining). Mean ± SD (three repeats) **** P < 0.0001; Student’s t -test. b Left: Heatmap comparing the gene expression profiles of KAL LU- and KAL LU+ cells by gene array analyses. Right: Some upregulated genes in KAL LU+ cells compared to KAL LU- cells. c Immunoblotting analysis verifying the expression of listed genes from Fig. 2b in KAL LU- and KAL LU+ cells. β-actin, a protein-loading control. d Comparison of KAL LU- and KAL LU+ cell proliferation using the MTT assay. Mean ± SEM (three repeats). ** P < 0.01; Student’s t -test. e Tumor burden in the lungs generated by KAL LU- and KAL LU+ cells in KA/KA mice ( n = 5/group). Mean ± SD; *** P < 0.001; Student’s t -test. f Representative images of a well-differentiated lung SCC generated by KAL LU- cells and spindle cell carcinoma generated by KAL LU+ cells in the lungs of KA/KA mice with the histological morphology stained with hematoxylin and eosin (H&E). Scale bar: 50 μM. g IHC staining of SCCs obtained from tail-vein injected KAL LU- and KAL LU+ cells with anti-K5, anti-involucrin, and anti-loricrin antibodies. Scale bar: 40 μM. h Survival curves of patients with lung SCCs expressing high TNFRSF1A or low TNFRSF1A levels (left), double-high TNFRSF1A and TWIST1 or double-low TNFRSF1A and TWIST1 levels (middle); and double-high TNFRSF1A and CDK6 or low TNFRSF1A levels (right). These data were obtained from OncoLnc.

Article Snippet: A mouse TNFR1 full-length cDNA fragment (1,365 bp) in the CMV-Myc Flag TNFR1 vector (MR226545, OriGene, Rockville, MD) was purified after being digested with KpnI and PmeI .

Techniques: Western Blot, Control, Flow Cytometry, Expressing, Fluorescence, Staining, Gene Expression, Comparison, MTT Assay, Generated, Immunohistochemistry, Injection

Journal: Cell Death & Disease

Article Title: A TNFR1–UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop

doi: 10.1038/s41419-022-05308-4

Figure Lengend Snippet: a Immunoblotting analysis showing TNFR1 levels in KAL LU+ cells treated with TNFR1 siRNA (si-TNFR1) or si-Control. β-actin, a protein-loading control. b Growth curve of KAL LU+ cells treated with si-TNFR1 or si-Control. Mean ± SD (three repeats); *** P < 0.001; Two-way AVOVA statistical test. c Immunoblotting analysis shows indicated protein levels in KAL LU+ cells treated with si-Control (si-Con) or si-TNFR1. β-actin, a protein-loading control. d , e Comparing tumor sizes ( d ) and numbers ( e ) derived from KAL LU+ cells treated with si-Control (si-Con) or si-TNFR1 in WT and KA/KA mice (recipient, n = 5/group). Each symbol represents a tumor. Mean ± SD; *** P < 0.001; Student’s t -test. f H&E-stained and E-cadherin IHC-stained sections of tumors derived from KAL LU+ cells treated with si-Control or si-TNFR1 in KA/KA mice (recipient). Scale bar: 40 μM. g K5 IHC staining for lung SCCs derived from si-TNFR1 KAL LU+ cells in KA/KA mice at 1.5 months after cell injections. Scale bar: 50 μM. h Immunoblotting analysis showing the levels of K5 and TNFR1 in KAL LU+ cells treated with si-Control and si-TNFR1. β-actin, a protein-loading control. i RT-PCR analysis showing TNF expression in WT and L-KA/KA lungs (left) and in M2C and KAL LU+ cells (right) treated with si-Control (si-Con) and si-TNFR1 ( n = 3/group). Mean ± SD (three repeats per group); *** P < 0.001; Student’s t -test. j , k Comparing tumor sizes ( j ) and tumor numbers ( k ) derived from KAL LU+ cells treated with si-Control (si-Con) or si-TNF in WT and KA/KA mice ( n = 5/group). Mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001; Student’s t -test. l Weights of tumors isolated from nude mice receiving subcutaneous injections of KAL LU+ cells treated with si-Control (si-Cont) and si-TNF RNA ( n = 5/group). Mean ± SD; ** P < 0.01; Student’s t -test.

Article Snippet: A mouse TNFR1 full-length cDNA fragment (1,365 bp) in the CMV-Myc Flag TNFR1 vector (MR226545, OriGene, Rockville, MD) was purified after being digested with KpnI and PmeI .

Techniques: Western Blot, Control, Derivative Assay, Staining, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation

Journal: Cell Death & Disease

Article Title: A TNFR1–UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop

doi: 10.1038/s41419-022-05308-4

Figure Lengend Snippet: a RT-PCR showing UBCH10 expression in KAL LU+ cells treated with si-Control (si-Cont) and si-TNFR1. *** P < 0.001; Student’s t -test (mean ± SD of three samples per group). b Left: Immunoblotting analysis showing levels of the indicated proteins in KAL LU+ cells treated with si-Control (si-Con) and si-UBCH10 (si-UB). β-actin, a protein-loading control. Right: Immunoblotting analysis showing Twist and UBCH10 levels in KAL LU+ (-), si-TNFR1 RNA-treated KAL LU+ cells (si-TNFR1 RNA including si-A, si-B, si-C, and si-ABC), and si-TNFR1 RNA-treated KAL LU+ cells with reintroduced Ube2c/UBCH10 cDNA. β-actin, a protein-loading control. c Left: Comparison the growth of tumors, derived from KAL LU+ cells treated with si-Control or si-UBCH10, in nude mice ( n = 5/group). Mean ± SD; * P < 0.01; two-way AVOVA test. d Survival curves of lung SCC patients with high TNFRSF1A and UBE2C levels or low TNFRSF1A levels. Lung SCC patient cohorts were obtained from Kaplan–Meier plotter. e Left: Immunoblotting analysis showing levels of K5, E-cadherin, and UBCH10 in KAL LU+ cells overexpressing a control or HA-IKKα vector. β-actin, a protein-loading control. Right: RT-PCR examines TNF expression in KAL LU+ cells and KAL LU+ cells overexpressing HA-IKKα with. Mean ± SEM (three repeats per group). ** P < 0.01; Student’s t -test. f Growth curves of KAL LU+ cells that were transfected with a control vector or HA-IKKα vector ( n = 5/group). Mean ± SD; ** P < 0.01; two-way AVOVA statistical test. g The sizes of tumors obtained from KAL LU+ cells treated with the control or HA-IKKα vector (2 × 10 5 cells per mouse) were subcutaneously injected into nude mice ( n = 5/group). Tumor growth was analyzed by a grouped two-way AVOVA statistical test; Mean ± SD; * P < 0.05. h Left: Images showing a liver (top) and lung (bottom) with metastases (Fig. shows H&E histological images). Right: H&E images of liver and lung metastases induced by arrows. Scale bar: 40 μM. i RT-PCR showing UBCH10 mRNA levels in KAL LU+ cells transfected with IKKα or control vectors ( n = 3/group). Mean ± SD; *** P < 0.001; Student’s t -test. j A working model for the TNFR1-UBCH10 pathway predicted to increase c-Myc, Bcl2, Twist1, and Sox2 expression and SCC dedifferentiation. IKKα inhibits UBCH10 expression. Arrow, enhancement; two lines, inhibition. k Human H520 SCC cell line was selected with stem cell markers and used to generate two sub-populations (left). H520 (CD24 lo CD44 + ) cells express elevated TNFR1 levels compared to H520 (CD24 hi CD44 + ) cells. Mean ± SD (three repeats per group); *** P < 0.001; Student’s t -test. l H520 (CD24 lo CD44 + ) cells generated tumors induced by subcutaneous injections and metastasis to lungs in nude mice ( n = 5). Scale bar: 40 μM.

Article Snippet: A mouse TNFR1 full-length cDNA fragment (1,365 bp) in the CMV-Myc Flag TNFR1 vector (MR226545, OriGene, Rockville, MD) was purified after being digested with KpnI and PmeI .

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, Comparison, Derivative Assay, Plasmid Preparation, Transfection, Injection, Inhibition, Generated

Journal: Cell Death & Disease

Article Title: A TNFR1–UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop

doi: 10.1038/s41419-022-05308-4

Figure Lengend Snippet: a Immunoblotting showing levels of the indicated nuclear and cytoplasmic protein levels in KAL LU+ and KAL LU- cells following TNF (10 ng/mL) stimulation. α-Tubulin, a cytosol protein-loading control. b Survival curves of human lung SCC expressing double-high TNFRSF1A and REL levels compared to low TNFRSF1A and REL levels. These data were obtained from the Kaplan–Meier plotter. c Immunoblotting showing UBCH10, E-cadherin, and keratin 5 (K5) levels in KAL LU+ cells treated with and without TNF (10 ng/mL) treatment for 60 min. β-actin, a protein-loading control. d RT-PCR shows UBCH10 expression in KAL LU+ cells treated with TNF (10 ng/mL). Mean ± SEM (three repeats per group). ** P < 0.01; Student’s t -test. e Immunoblotting showing levels of the indicated protein levels in KAL LU+ cells following treatment with three different si-UBE2C RNAs (A, B, C, and A + B + C). β-actin, a protein-loading control. f ChIP assay showing the enrichment of p65, c-Rel, and IKKα associated with the Ube2c promoter (p) on regions upstream of −500 bp and −1000 bp in KAL LU+ cells, immunoprecipitated with p65, c-Rel, and IKKα antibodies. The enrichment was compared to input levels. anti- or Ab, antibody; Neg-Ab, negative-control antibody (Ig); Pos-Ab, positive experimental anti-p65, c-Rel, and IKKα antibody; +IKKα, overexpressed HA-IKKα in KAL LU+ cells. g ChIP assay showing the enrichment of p65 associated with the Ube2c promoter (p) on regions upstream of −500 bp and −1000 bp, immunoprecipitated with p65 antibody in KAL LU- and KAL LU+ cells. The enrichment was compared to input levels. anti- or Ab, antibody; Neg-Ab, negative-control antibody; Pos-Ab, positive p65 antibody; TNF, TNF (10 ng/mL) treatment for 45 min. h ChIP assay showing the enrichment of H3K27ac on the Ube2c promoter (−500 bp) in KAL LU+ cells treated with and without TNF (10 ng/mL). i ChIP assay showing the enrichment of H3K27ac on the Tnfrsf1a promoter (−500 bp) in KAL LU- and KAL LU+ cells. j A working model for how overexpressed TNFR1 promotes and IKKα inhibits Ube2c gene expression, resulting in SCC cell dedifferentiation, aggressive tumors, and metastasis. Silencing TNFR1 or UBCH10 inhibits TNFR1-enhanced tumorigenesis. Arrow, promotion; crossed lines, inhibition.

Article Snippet: A mouse TNFR1 full-length cDNA fragment (1,365 bp) in the CMV-Myc Flag TNFR1 vector (MR226545, OriGene, Rockville, MD) was purified after being digested with KpnI and PmeI .

Techniques: Western Blot, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Gene Expression, Inhibition

Journal: bioRxiv

Article Title: Novel mechanism of inflammatory activation by Ebola virus matrix protein linked to the ebolavirus virulence

doi: 10.1101/2024.10.06.616882

Figure Lengend Snippet: a Diagram of the canonical and non-canonical NF-κB pathways. The canonical pathway involves the IκB kinase (IKK) complex, which comprises IKKα, IKKβ, and IKKγ. Activation of the IKK complex leads to the phosphorylation/ubiquitination and subsequent proteasomal degradation of Iκβα, allowing either p65:p50 or cRel:p50 NF-κB heterodimers to enter the nucleus. The non-canonical pathway relies on the cooperation of NF-κB-inducing kinase (NIK) and its downstream kinase IKKα. Activation of NIK and IKKα triggers the proteasomal processing of p100 into p52, leading to the nuclear translocation of RelB:p52 NF-κB heterodimers. b Western blotting for NF-κB subunits in cytoplasmic and nuclear fractions isolated from 293 cells expressing EBOV VP40 or TRAF6 at 24 hpt. c NF-κB-responsive luciferase reporter activity in 293 cells expressing EBOV VP40 at 48 hpt, following siRNA-mediated knockdown of p65 or GAPDH. d Western blotting for IKK complex and Iκβα in 293 cells expressing EBOV VP40, EBOV GP, or TRAF6 at 24 hpt. e NF-κB-responsive luciferase reporter activity in 293 cells expressing 0.25 μg of IKK wild-type or IKK mutant (K44M) together with either 0.25 μg of EBOV VP40 or TRAF6 at 48 hpt. f NF-κB-responsive luciferase reporter activity in Iκβα-knockout 293 cells expressing 0.25 μg of Iκβα wild-type or Iκβα mutant (SS32/36AA) together with 0.25 μg of EBOV VP40 at 48 hpt. g NF-κB-responsive luciferase reporter activity in 293 cells expressing 0.01, 0.1, or 0.5 μg of Iκβα together with 0.5 μg of EBOV VP40 at 48 hpt. h Western blotting for p65 in cytoplasmic and nuclear fractions isolated from 293 cells expressing EBOV VP40 with or without Iκβα, TRAF6, or Iκβα at 24 hpt. Control: Empty vector transfected. For c and e - g , data are shown with mean ± SD (n = 3 independent biological replicates). ns > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001; ordinary one-way ANOVA.

Article Snippet: The following open reading frames were cloned into an expression vector under the control of the phosphoglycerate kinase promoter: IKK-2 and its mutant IKK-2 K44M (Addgene plasmids #11103 and #11104, gifts from Anjana Rao ); 3xHA-Iκβα and its mutants 3xHA-Iκβα-SS32/36AA (Addgene plasmids #21985 and #24143, gifts from Warner Greene ); TNFR1-YFP (Addgene plasmid #111209, gift from Johannes A. Schmid).

Techniques: Activation Assay, Translocation Assay, Western Blot, Isolation, Expressing, Luciferase, Activity Assay, Knockdown, Mutagenesis, Knock-Out, Control, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Novel mechanism of inflammatory activation by Ebola virus matrix protein linked to the ebolavirus virulence

doi: 10.1101/2024.10.06.616882

Figure Lengend Snippet: a Western blotting for molecules involved in the non-canonical NF-κB pathway. 293 cells were transfected with plasmid EBOV VP40, EBOV GP, TRAF6, or NIK and harvested at 24 hpt or treated with 15 ng/ml of TNFα for 1 hour and then harvested. b Western blotting for phosphorylated-Iκβα or Iκβα in 293 cells expressing EBOV VP40 or TRAF6. c Quantification of Iκβα mRNA in EBOV VP40-expressing 293 cells. Extracted RNA was reverse transcribed using OligodT primer, and cDNA was used for qRT-PCR with iTaq Universal Probes Supermix (BioRad), Hs00355671_g1 NFKBIA (Thermo Scientific) and Hs00355671_g1 NFKBIA (Thermo Scientific). GAPDH was used as a reference gene (the primer/TaqMan probe sequences are shown in Supplementary Table 3) . Delta-delta C t values were used to determine their relative expression as fold changes. Details on RNA preparation are provided in the Materials and Methods section. d Western blotting for phosphorylated-Iκβα or Iκβα in 293 cells expressing EBOV VP40, EBOV GP, TRAF6, NIK or RESTV VP40 at 24 hpt. e Western blotting for phosphorylated-Iκβα or Iκβα in 293 cells expressing EBOV VP40, EBOV VP40 Δ1-20aa , or chimeric VP40 proteins where the HVR of EBOV VP40 was replaced with that of RESTV or BDBV (EVP40 HVR-RESTV and EVP40 HVR-BDBV ) at 24 hpt. Control: Empty vector transfected. For c , data are shown with mean ± SD (n = 3 independent biological replicates).

Article Snippet: The following open reading frames were cloned into an expression vector under the control of the phosphoglycerate kinase promoter: IKK-2 and its mutant IKK-2 K44M (Addgene plasmids #11103 and #11104, gifts from Anjana Rao ); 3xHA-Iκβα and its mutants 3xHA-Iκβα-SS32/36AA (Addgene plasmids #21985 and #24143, gifts from Warner Greene ); TNFR1-YFP (Addgene plasmid #111209, gift from Johannes A. Schmid).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Reverse Transcription, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Novel mechanism of inflammatory activation by Ebola virus matrix protein linked to the ebolavirus virulence

doi: 10.1101/2024.10.06.616882

Figure Lengend Snippet: a Synthego ICE analysis for CRISPR editing targeting a AAVS1, b TNFR1, c LTβR, d TNFα. e Western blotting for Iκβα in Iκβα-knockout 293 and wild-type 293 cells.

Article Snippet: The following open reading frames were cloned into an expression vector under the control of the phosphoglycerate kinase promoter: IKK-2 and its mutant IKK-2 K44M (Addgene plasmids #11103 and #11104, gifts from Anjana Rao ); 3xHA-Iκβα and its mutants 3xHA-Iκβα-SS32/36AA (Addgene plasmids #21985 and #24143, gifts from Warner Greene ); TNFR1-YFP (Addgene plasmid #111209, gift from Johannes A. Schmid).

Techniques: CRISPR, Western Blot, Knock-Out

Journal: Molecular and Cellular Biology

Article Title: Autocrine Tumor Necrosis Factor Alpha Links Endoplasmic Reticulum Stress to the Membrane Death Receptor Pathway through IRE1α-Mediated NF-κB Activation and Down-Regulation of TRAF2 Expression

doi: 10.1128/mcb.26.8.3071-3084.2006

Figure Lengend Snippet: FIG. 5. ER stress-induced NF-B activation is independent of TNFR1 signaling. (A) ER stress-induced NF-B activation does not re- quire de novo protein synthesis. MCF-7 cells were treated with or without CHX (5 M) for 1 h and then with thapsigargin or tunicamycin for 2 h. Nuclear extracts were prepared for EMSA with an NF-B probe. (B) Knockdown of TNFR1 by siRNA. MCF-7 cells were transfected with a control siRNA and a specific siRNA pool to TNFR1. Forty-eight hours after transfection, cell lysates were subjected to Western blotting with anti-TNFR1 and anti--actin antibodies. (C) Impaired TNF--induced NF-B activation by ablation of TNFR1. After transfection with TNFR1- siRNA and control siRNA (ContsiRNA) for 48 h, MCF-7 cells were subjected to TNF- (15 ng/ml) for 1 and 2 h. Cell extracts were collected and used to analyze protein levels of IB and -actin by Western blotting with the indicated antibodies. (D and E) ER stress-induced NF-B is independent of TNFR1 signaling. MCF-7 cells were transfected with the indicated siRNAs as described for panel C. After treatment with thapsi- gargin or tunicamycin for the indicated times, whole-cell extracts and nuclear extracts were prepared. Cell extracts from each sample were analyzed for IB levels by Western blotting. Nuclear extracts were ex- amined by EMSA with an NF-B probe. TG, thapsigargin; TU, tunica- mycin; Conti, control siRNA.

Article Snippet: The following siRNAs were used: siRNA pool to human IRE1 , siRNA pool to human TNFR1, a control siRNA (all from Dharmacon), siRNA to human RelA (Upstate), and siRNA to human caspase-8 (Molecula).

Techniques: Activation Assay, Knockdown, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Autocrine Tumor Necrosis Factor Alpha Links Endoplasmic Reticulum Stress to the Membrane Death Receptor Pathway through IRE1α-Mediated NF-κB Activation and Down-Regulation of TRAF2 Expression

doi: 10.1128/mcb.26.8.3071-3084.2006

Figure Lengend Snippet: FIG. 6. Autocrine TNF- contributes to NF-B-mediated cell death in ER stress. (A) TNFR1-Fc decreases ER stress-induced cell death. MCF-7 cells were exposed to thapsigargin or tunicamycin with or without TNFR1-Fc at different concentrations for 30 h. Cell death was detected by MTT assay. Data are representative of triplicate experiments. (B) TNFR1-Fc does not affect camptothecin-induced cell death. MCF-7 cells were incubated with camptothecin (1 M) with or without TNFR1-Fc for 24 h. Cell death was detected by MTT assay as described for panel A. (C) Fas-Fc does not influence ER stress-induced cell death. MCF-7 cells were treated with thapsigargin or tunicamycin in the presence or absence of Fas-Fc for 30 h. Cell death was detected by MTT assay as described for panel A. (D) Elimination of TNFR1 inhibits ER stress-induced cell death. After transfection with TNFR1-siRNA and control siRNA (ContsiRNA) for 48 h, MCF-7 cells were exposed to thapsigargin, tunicamycin, or camptothecin (1 M) for indicated times. Cell death was scored by MTT assay as described for panel A. Cell survival was expressed as absorbance relative to that of DMSO-treated controls. TG, thapsigargin; TU, tunicamycin; Campt, camptothecin.

Article Snippet: The following siRNAs were used: siRNA pool to human IRE1 , siRNA pool to human TNFR1, a control siRNA (all from Dharmacon), siRNA to human RelA (Upstate), and siRNA to human caspase-8 (Molecula).

Techniques: MTT Assay, Incubation, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: Autocrine Tumor Necrosis Factor Alpha Links Endoplasmic Reticulum Stress to the Membrane Death Receptor Pathway through IRE1α-Mediated NF-κB Activation and Down-Regulation of TRAF2 Expression

doi: 10.1128/mcb.26.8.3071-3084.2006

Figure Lengend Snippet: FIG. 7. ER stress sensitizes cells to TNF--induced cell death. (A) MCF-7 cells were treated with or without thapsigargin or tunicamycin for 4 h. After being washed with medium (Dulbecco’s modified Eagle medium 10% FBS) two times, cells were incubated without or with TNF- (30 ng/ml). Cell death was quantified by MTT assay. Data are representative of triplicate experiments. (B) ER stress inhibits TNF--induced activation of NF-B. MCF-7, L929, and DU145 cells were treated with DMSO, thapsigargin, or tunicamycin for 4 h and then stimulated with TNF- (15 ng/ml) for the indicated times. Cell lysates were prepared, and 20 g of protein from each sample was used for Western blotting with anti-IB and anti--actin. (C) ER stress induces degradation of TRAF2. MCF-7 and L929 cells were treated with thapsigargin or tunicamycin. Cell lysates were prepared, and 20 g of protein from each sample was used for Western blotting with anti-TNFR1, anti-TRADD, anti-RIP, anti-TRAF2, anti-IKK, and anti-IKK. (D) ER stress inhibits TNF--induced activation of JNK. The same samples from panel D were used for immunoblotting with anti-JNK and anti-phospho-JNK antibodies. (E) ER stress does not influence transcription of TRAF2. MCF-7 cells were treated with thapsigargin or tunicamycin for 2 and 4 h. Total RNA was collected, and the mRNA level of TRAF2 was measured by RT-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) ER stress decreases stability of TRAF2. L929 cells were incubated with CHX (20 M) or CHX plus thapsigargin (2 M) for the indicated time, and then the TRAF2 protein level was examined by Western blotting. (G) Knockdown of TRAF2 protein sensitizes cells to ER stress-induced cell death. After transfection with TRAF2 siRNA or control siRNA for 48 h, MCF-7 cells were subjected to thapsigargin or tunicamycin for the indicated times. Cell death was assessed by an MTT assay. TG, thapsigargin; TU, tunicamycin; Conti, control siRNA.

Article Snippet: The following siRNAs were used: siRNA pool to human IRE1 , siRNA pool to human TNFR1, a control siRNA (all from Dharmacon), siRNA to human RelA (Upstate), and siRNA to human caspase-8 (Molecula).

Techniques: Incubation, MTT Assay, Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Knockdown, Transfection, Control