Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 1 Effect of IL1B on mbIL1R, mbIL1R2 and IL1RA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A) or with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (B). Cells were recovered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by western blot. a-Tubulin was also probed on the same membranes to ensure equal protein loading. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (C and D). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data are from three different experiments. MM, minimal medium; mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Control, Incubation, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 3 Immunocytofluorescence of IL1R1, IL1R2 and IL1RA in KLE cells. Cells cultured in chamber slides were incubated for 24 h with the culture medium alone or containing 0.1 ng/ml IL1B. Detection of IL1R1 (A and C), IL1R2 (E and G) and IL1RA (I and K) was performed by immunocytochemical staining using specific monoclonal mouse antibodies. Note the increase in IL1R1 (C), IL1R2 (G) and IL1RA (K) immunofluorescent signal in KLE cells exposed to IL1B by comparison with non-stimulated cells (A, E and I, respectively). No immunofluorescence was observed in the absence of primary antibodies (B, F and J) or the presence of mouse IgGs (D, H and L) (controls). Data are representative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Cell Culture, Incubation, Staining, Comparison

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 4 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A, B and C, respectively) or with different con- centrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (D, E and F, respectively). Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1, IL1R2 and IL1RA mRNA levels were normalized to GAPDH mRNA levels. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of time). *P , 0.05, **P , 0.01 and ***P , 0.001 as compared with control. Data are from three different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 5 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA stability and gene expression in KLE cells. Confluent KLE cell cultures were stimulated with IL1B (1 ng/mll) for 12 h. Actinomycin D (10 mg/ml) was added to stop the de novo RNA synthesis, and cells were harvested after 0, 2, 4 and 8 h of incubation with actinomycin D. Total RNA was extracted and reverse transcribed. cDNA was analyzed by real-time PCR with specific primers for IL1R1, IL1R2, IL1RA and GAPDH. Levels of IL1R1, IL1R2 and IL1RA mRNA were normalized to those of GAPDH to assess the kinetics of IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA degradation. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found at different periods of time following the arrest of de novo mRNA transcription to that found at the time of arrest); data are from three different experiments. To evaluate IL1R1, IL1R2 and IL1RA transcriptional activation (D), confluent KLE cell cultures were stimulated with IL1B (1 ng/ml) for 6 h, cell nuclei were isolated and nuclear mRNA transcription was analyzed by nuclear run-on as described in Materials and Methods. DNA samples immobilized onto nylon membranes were as follows: lane 1, IL1R1 cDNA; lane 2, IL1R2 cDNA; lane 3, IL1RA; lane 4, 28S cDNA; and lane 5, pBluescript plasmid DNA. Radioactive transcripts were from KLE cells stimulated in MM or with 1 ng/ml IL1B. Data are repre- sentative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Gene Expression, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Activation Assay, Isolation, Plasmid Preparation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 6 Effect of IL1B on mbIL1R1, mbIL1R2 and IL1RA expression in endometrial epithelial cells. Confluent endometrial epithelial cell cultures were treated with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Cells were recov- ered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by Western blot (A). a-Tubulin was probed on the same membranes to ensure equal protein loading. Culture supernatants were recovered to evaluate sIL1R2 release (C) and IL1RA secretion (D) by ELISA; data were from four different endometrial samples and expressed as % of control (ratio of sIL1R2 or IL1RA concentrations detected in the presence of IL1 to those detected in the control basal culture medium for an equivalent period of 12 h). *P , 0.05, as compared with control. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (B). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data were from three different endometrial samples. sIL1R1 and sIL1R2 release and IL1RA secretion from endometrial epithelial cell cultures was also analyzed by metabolic labeling with 35S-cysteine and immunoprecipitation using a goat anti-IL1R1, goat anti-IL1R2 or rabbit anti- IL1RA antibody (E). Immunoprecipitated proteins were then analyzed by SDS-PAGE as described in Materials and Methods (representative data from three different endometrial samples); mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Labeling, Immunoprecipitation, SDS Page, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 7 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in endometrial epithelial cells. Confluent cultures were treated with 0.1 ng/ml IL1B with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA levels were normalized to GAPDH mRNA levels. Data were from three different endometrial samples and expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of 12 h). *P , 0.05 and **P , 0.01 as compared with control.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Schematic of the urea cycle. b , Related metabolites in BMDMs treated with VSV for 6 h or not (NT), assayed by LC–MS/MS. log 2 fold changes are relative to NT. Citrulline, argininosuccinate, fumarate, aspartic acid, putrescine and glutamine increased significantly ( P < 0.05, two-tailed Student’s t -test) ( n = 3). c , Heatmap of gene expression in BMDMs treated with VSV for 6 h or not (NT), assessed by qPCR. log 2 fold changes are relative to NT. SMS , SAT1 , SMOX , ASS1 , IDH3b and CS increased significantly ( P < 0.05, two-tailed unpaired Student’s t -test) ( n = 3). d , IFNβ expression in BMDMs pretreated with indicated metabolites overnight and infected with VSV for 6 h or not (NT), assessed by qPCR ( n = 3). e , IFNβ expression in BMDMs pretreated with DMF (5 µM), MMF (20 µM), FHINI (20 µM) or DMSO (Ctrl) overnight, followed by VSV infection for 6 h or not (NT), determined by qPCR ( n = 3). f , Ki67 + BMDM percentage (left) and survival rate (right) after culture in fumarate (2 mM), DMF (5 µM) or MMF (20 µM) overnight, measured by FACS ( n = 3). g , IFNβ levels in BMDM supernatants pretreated overnight with DMF (5 µM) or DMSO followed by VSV infection for 6 h or not (NT), determined by ELISA ( n = 3). h , VSV transcript in BMDMs pretreated overnight with DMF (5 µM) or DMSO (−) followed by VSV (+) infection for 6 h, analysed by qPCR ( n = 3). i – n , Mice treated with DMF or DMSO for 7 days before the VSV challenge. Serum fumarate levels ( i ; n = 3) and IFNβ mRNA ( j ), serum IFNβ protein ( k ), VSV mRNA ( l ), lung histology ( m ), and ASS1 and IFNβ expression in peritoneal macrophages ( n ) were assessed ( j – n ; n = 5). The scale bar (200 µm) applies to all images in m . Statistical analysis: two-tailed Student’s t -test ( b , c , f , h and i ) or two-way ANOVA ( d , e , g , j , k , l and n ). Data are the mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. CPS1, carbamoyl phosphate synthetase 1; OTC, ornithine transcarbamylase; ASL, argininosuccinate lyase; ARG1, arginase 1; ODC, ornithine decarboxylase; ASS1, argininosuccinate synthase.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Gene Expression, Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Heatmap of gene expression in VSV-infected BMDMs. Log2 fold changes are relative to uninfected BMDMs (0 h) (n = 3). b - c , IFN-β transcripts in iBMDMs pretreated with metabolites, and then infected with VSV for 6 h or not (NT) (n = 3). d , VSV plaque morphology in Vero cells after DMF (20 μM) or DMSO treatment for 6 h (n = 4). e - g , iBMDMs pretreated with DMF (5 µM) or DMSO, followed by VSV infection for 6 h or not (NT). IFN-β and ASS1 transcripts ( e ), VSV expression ( f ), and IFN-β protein ( g ) (n = 3). h , ASS1 enzymatic activity in BMDMs infected with VSV for 6 h or not (−) (n = 3). i , Enzymatic activity of ASS1, purified from HEK293 cells infected with VSV or not, was analyzed (n = 3). j , Expression of OTC, ASS1, ASL and ARG1 in BMDMs treated with VSV or not (−), measured by Western blot. k , NO levels in ASS1 +/+ and ASS1 −/− BMDMs infected with VSV or not (−) (n = 3). l-n , ASS1 transcripts in BMDMs treated with IFN-β (10 pg/ml) for different time points ( l ), treated with increasing concentrations of IFN-β for 6 h ( m ), and infected with VSV or not in the presence of IFN-β ( n ) (n = 3). o , IFN-β protein levels in supernatants of BMDMs infected with VSV or not (−), determined by ELISA (n = 3). p , ASS1 expression in BMDMs infected with VSV or not (−) in the presence of IFN-β (100 pg/ml) or not (−) for 6 h, measured by qPCR (n = 3). Statistical analysis: two-tailed Student’s t -test ( a , d , f , h , i, l , m , o and p ) or two-way ANOVA ( b , c, e, g and k ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Gene Expression, Infection, Expressing, Activity Assay, Purification, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Fumarate, malate and aspartate levels in BMDMs derived from macrophage-specific ASS1 knockout ( ASS1 −/− ) mice and corresponding wild-type ( ASS1 +/+ ) mice pretreated with AOAA (5 mM) or not (−), followed by VSV infection for 6 h or not (−), measured by LC–MS/MS/MS ( n = 3). b , Atom-transition map showing that the isotope carbon-13 ( 13 C) transfers from [U- 13 C 6 ]glutamine through the AAS shunt. The open circles represent carbon-12 ( 12 C); the green circles indicate 13 C from [U- 13 C 6 ]glutamine. c – e , Incorporation of 13 C atoms into fumarate ( c ), aspartate ( d ) and malate ( e ) in BMDMs from ASS1 +/+ and ASS1 −/− mice cultured with [U- 13 C 6 ]glutamine and treated with VSV or not (−) for 6 h, determined by LC–MS/MS ( n = 3). f , g , Expression of ASS1, IFNβ and VSV ( f ) in BMDMs pretreated overnight with DMF (5 µM) or DMSO (−) followed by VSV infection for 6 h or not (−), determined by qPCR and IFNβ protein levels by ELISA ( g ) ( n = 3). h , OCR analysis of BMDMs from ASS1 +/+ and ASS1 −/− mice infected with VSV for 6 h or not (−). Basal respiration, maximal respiration, proton leak and reserve respiratory capacity were analysed ( n = 4). i , Schematic illustration of the respiratory chain. j , Expression of respiratory chain genes in BMDMs infected with VSV for 6 h or not (NT), analysed by qPCR. log 2 fold changes are relative to BMDMs not infected with VSV (NT). Significant decreases in NDUFA1 , NDUFA3 , NDUFA4 and others ( n = 3). k , OCR analysis of ASS1 +/+ ( n = 4) and ASS1 −/− ( n = 3) BMDMs treated with VSV infection for 6 h or not (−) in the presence or absence of 1 mM succinate. Statistical analysis: two-tailed Student’s t -test ( a and j ) or two-way ANOVA ( c – g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. CoQ, coenzyme Q; Cyt c, cytochrome complex; Suc, Succinate. Panel i created with BioRender.com .

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Knock-Out, Infection, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a - e , The incorporation of 13 C atoms into α-ketoglutarate ( a ), succinate ( b ), isocitrate ( c ), aconitate ( d ) and citrate ( e ) in BMDMs from ASS1 +/+ and ASS1 −/− mice cultured in medium containing 2 mM [U- 13 C 6 ]-glutamine and treated with VSV or not (−) for 6 h was determined by LC-MS/MS. The isotopic labeling of each metabolite is denoted as m + n, where n is the number of 13 C atoms (n = 3). f , Luciferase assay analysis of IFN-β promoter activity in HEK293 cells transfected with control siRNA (siCtrl) or ASS1 siRNA (siASS1) and then treated with VSV for a further 6 h (n = 3). Protein expression was analyzed by Western blot. g , HEK293 cells transfected with an increasing amount of Flag-tagged ASS1 plasmids for 24 h. The expression of IFN-β (left) and ASS1 (right) was determined by qPCR (n = 3). h , IFN-β transcript in BMDMs pretreated with DMF (5 µM) overnight, then followed by VSV infection for 6 h in the presence or absence of AOAA (5 mM) was determined by qPCR (n = 3). i , ASS1 +/+ and ASS1 −/− BMDMs treated with VSV for 6 h or not (−) were used for OCR analysis (n = 4). Relative to Fig. . j , IFN-β expression in BMDMs treated with DECA (10 µM) or not (PBS) followed by VSV infection for 6 h was analyzed by qPCR (n = 3). k , ASS1 +/+ (n = 4) and ASS1 −/− (n = 3) BMDMs treated with VSV or not (−) for 6 h in the presence or absence of 1 mM succinate were used for OCR analysis. Relative to Fig. . Statistical analysis: two-tailed Student’s t -test ( f - h ) or two-way ANOVA ( a-e and i-k ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Isotopic Labeling, Luciferase, Activity Assay, Transfection, Control, Expressing, Western Blot, Infection, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Luciferase reporter assays for IFNβ promoter activity in HEK293 cells transfected with indicated vectors, treated with DMF (10 µM) or DMSO for 4 h, followed by measurement of IFNβ promoter activity and western blot analysis ( n = 3). b , ASS1 +/+ and ASS1 −/− BMDMs pretreated with DMF (5 µM) or DMSO (−) overnight, followed by VSV infection for 6 h or not (−), analysed by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with VSV for 6 h or not (−), and MAVS succination was assessed by immunoblotting. d , e , Immunoprecipitated Flag-MAVS from HEK293 cells treated with DMF (10 μM) for 6 h was subjected to mass spectrometry for MAVS succination. Cysteine residues at positions 46 ( d ) and 283 ( e ) in MAVS are susceptible to succination by fumarate. f , HEK293 cells transfected with Flag-MAVS or mutants, treated with 10 μM DMF for 6 h and analysed for MAVS succination by immunoblotting. g , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) were treated with VSV for 6 h or not (−) and analysed for MAVS succination. h , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) treated VSV for 6 h and IFNβ and VSV expression analysed by qPCR (left) ( n = 3). Protein expression measured by western blot (right). i , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) treated with DMF (5 μM) or not for 6 h and analysed for MAVS succination by immunoblotting. j – m , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) were pretreated with DMF (5 μM) or not (−), followed by VSV infection for 6 h. IFNβ, MAVS and VSV transcripts were determined by qPCR ( j ) ( n = 3). Cell lysates were analysed by SDS–PAGE ( k and m ), and MAVS aggregation was examined by SDD–AGE ( l ). Statistical analysis: two-tailed Student’s t -test ( a and h , right) or two-way ANOVA ( h , left, and j ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. Luc, luciferase; WB, western blot.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Infection, Immunoprecipitation, Mass Spectrometry, Transduction, Mutagenesis, Expressing, SDS Page, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Protein expression of BMDMs pretreated with DMF (5 μM) overnight or not (−), followed by VSV infection, was determined by Western blot. b , Protein expression in BMDMs pretreated overnight with the indicated metabolites followed by VSV infection or not (−) was determined by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with MMF (20 μM), DMF (10 μM or 20 μM), fumarate (200 μM) respectively or not (−) for 6 h. MAVS succination was analyzed by immunoblotting (left), and densitometry quantified the succinated-to-total MAVS ratio (right). d , Schematic of RIG-I-like Receptor (RLR) signaling. e , MAVS-deficient iBMDMs transduced with human MAVS were transfected with siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection and DMF (5 μM) treatment for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. f , MAVS-deficient iBMDMs transduced with human MAVS were transfected with both siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection or not (−) for 6 h. Whole cell lysates were analyzed by immunoblotting. g , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS (2CS) were transfected with siRNAs targeting RIG-I and MDA5, or vector control (siCtrl, -), followed by VSV infection for 6 h. IFN-β expression(left) and protein levels were measured (n = 3). h , Purified Flag-MAVS protein from HEK293 cells was used in in vitro assays to assess succination and aggregation of MAVS in the presence or absence of fumarate (1 mM) for 6 h. i , Purified Flag-MAVS and MAVS (2CS) proteins from HEK293 cells were used in in vitro assays to assess succination and aggregation of MAVS. Statistical analysis: two-tailed Student’s t -test ( c right) or two-way ANOVA ( g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Western Blot, Transfection, Transduction, Plasmid Preparation, Control, Mutagenesis, Purification, In Vitro, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a - b , ASS1 +/+ and ASS1 −/− BMDMs were infected with SeV or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and SeV in were analyzed by qPCR, and fumarate levels were analysed by LC-MS/MS ( a ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( b ). c - d , ASS1 +/+ and ASS1 −/− BMDMs were infected with IAV or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and IAV in were analyzed by qPCR, and fumarate levels were analyzed by LC-MS/MS ( c ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( d ). e - f , ASS1 +/+ and ASS1 −/− BMDMs were infected with HSV-1 or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and HSV-1 were analyzed by qPCR, and fumarate levels were analyzed by LC-MS/MS ( e ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( f ). g , Lysates from ASS1 +/+ and ASS1 −/− BMDMs infected with SeV (left), IAV (middle), or HSV-1 (right) or left uninfected (−) for 6 h was analyzed by western blot. h , IFN-β expression in MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS(2CS) infected for 6 h with SeV (left), IAV (middle), or HSV-1 (right) or left uninfected (−) was measured by qPCR (n = 3). i , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS(2CS) were infected with SeV, IAV, or HSV-1 or left uninfected (−) for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. Statistical analysis: two-tailed Student’s t -test (the fourth section of a , c and e ) or two-way ANOVA (the first three part of a , c and e , and h ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Transduction, Mutagenesis, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Expression of IFNβ in hMDMs expressing sgRNA-Ctrl ( ASS1 +/+ hMDMs) and hMDMs expressing sgRNA-ASS1 ( ASS1 −/− hMDMs) not infected or infected with VSV (left) or SeV in the presence or absence of DMF (10 µM) for 6 h was determined by qPCR analysis ( n = 3). b , Expression of VSV in ASS1 +/+ and ASS1 −/− hMDMs infected with VSV or SeV in the presence or absence of 10 µM DMF for 6 h was measured by qPCR analysis ( n = 3). c , Fumarate levels in ASS1 +/+ and ASS1 −/− hMDMs infected for 6 h with VSV or SeV, or uninfected (NT), were measured by LC–MS analysis ( n = 3). d , ASS1 +/+ and ASS1 −/− hMDMs in the presence or absence of 10 µM DMF were infected with VSV or left uninfected (−) for 6 h. Whole-cell lysates and anti-MAVS immunoprecipitants were analysed by immunoblotting. e , Whole lysates and immunoprecipitants with anti-MAVS from ASS1 +/+ and ASS1 −/− hMDMs in the presence or absence of DMF (10 µM) infected with SeV or not (−) were analysed by western blot. Statistical analysis: two-way ANOVA ( a – c ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Lysates from ASS1 +/+ and ASS1 −/− hMDMs infected with VSV or SeV or not (−) for 6 h in the presence or absence of DMF (10 µM) were analyzed by Western blot. b , ASS1 enzymatic activity in hMDMs treated with MDLA (10 mM) or not (−) for 6 h (left). Protein expression was also determined by immunoblotting (right) (n = 3). c , Fumarate levels in hMDMs treated with or without MDLA (10 mM) for 6 h was measured by LC-MS/MS (n = 3). d-f , hMDMs treated with MDLA (10 mM) or not (−) were infected with VSV ( d ), SeV ( e ), IAV ( f ), or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β, VSV, SeV and IAV were measured by qPCR respectively (n = 3). g , Lysates from hMDMs treated with MDLA (10 mM, +) or not (−) were infected with VSV, SeV, IAV, or left uninfected (−) were analyzed by Western blot. Statistical analysis: two-tailed Student’s t -test ( b-c ) or two-way ANOVA ( d-f ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Western Blot, Activity Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , b , In silico bioinformatic analysis of the expression of ASS1 using public databases. Expression of ASS1 was analysed between healthy people (Healthy) and patients infected with EVD using public databases ( GSE83565 ) ( a ). Box plots showing ASS1 expression in primary hMDMs treated with EBOV or MOCK, using data from the GSE84188 database ( b ). The box plot shows the minimum, maximum, median and interquartile range (IQR; 25th to 75th percentiles). Whiskers extend to 1.5 × IQR, and outliers are shown as individual points. c – g , ASS1 +/+ ( n = 5) and ASS1 −/− ( n = 5) mice were treated daily for seven consecutive days with 50 mg kg −1 DMF (dissolved in 10% DMSO) or 10% DMSO (−) by oral gavage, followed by intraperitoneal injection with VSV (2 × 10 7 PFU per mouse, +) or not (−). Mice were killed 6 h post-infection. c , d , mRNA levels of IFNβ ( c ) and VSV ( d ) in the lung and spleen were determined by qPCR analysis. e , Serum IFNβ protein levels were determined by ELISA analysis. f , IFNβ expression in splenic macrophage cells was analysed by qPCR (left, n = 3), and endogenous MAVS succination in splenic macrophage cells was measured by western blot (right). g , H&E staining of lung sections (scale bar, 100 μm). The scale bar applies to all images in g . h , Survival of ASS1 +/+ and ASS1 −/− mice intraperitoneally injected with VSV in the absence (VSV- ASS1 +/+ , n = 9; VSV -ASS1 −/− , n = 9) or presence of DMF dissolved in 10% DMSO (50 mg kg −1 , by oral gavage for seven consecutive days) (VSV, DMF- ASS1 +/+ , n = 10; VSV, DMF- ASS1 −/− , n = 12). i , Schematic illustrating the metabolic remodelling and upregulation of ASS1 to form the AAS cycle induced by viral infection to produce fumarate in the cytosol, promoting the antiviral response. Statistical analysis: two-tailed Student’s t -test ( a and b ), two-way ANOVA ( c – f ) or Kaplan–Meier survival analysis ( h ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. Arg, arginine; Orn, ornithine; Cit, citrulline; Asp, aspartate; Mal, malate; Suc, succinate. Panel i created with BioRender.com .

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Silico, Expressing, Infection, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , ASS1 +/+ and ASS1 −/− mice were intraperitoneally injected with VSV (2×10 7 pfu per mouse) or not (−). Mice were sacrificed 6 h post-infection. Endogenous MAVS succination was analyzed in the lungs of mice by Western blot. b , ASS1 +/+ and ASS1 −/− mice were treated daily for seven consecutive days with 50 mg/kg DMF (dissolved in 10% DMSO) or 10% DMSO (−) by oral gavage, followed by intraperitoneal injection with VSV (2 × 10 7 pfu per mouse) or not (−). Mice were sacrificed 6 h post-infection. Endogenous MAVS succination was analyzed in the lungs of mice by Western blot. c-j , ASS1 +/+ and ASS1 −/− BMDMs were infected with VSV for different time points as indicated. ASS1 transcripts ( c ), IFN-β protein levels ( d ), VSV ( e ) and IFN-β ( f ) transcripts, fumarate levels ( g ), phosphorylation of TBK1 and IRF3 ( h ), ASS1 activity ( i ), FH transcripts ( j ) were determined respectively (n = 3). k - l , Aspartate and citrulline levels in BMDM infected with VSV at different time points were measured by LC-MS/MS (n = 3). Statistical analysis: two-tailed Student’s t -test ( i, k and l ) or two-way ANOVA ( c-g and j ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Infection, Western Blot, Phospho-proteomics, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) SDS-PAGE migration of programmed membrane protein (proMP) C4.1 is consistent with a tetramer. Design model and peptide sequence shown for reference. ( b ) Rosetta ab initio structure prediction calculations predict that proMP C4.1 preferentially forms a tetramer. ( c ) CAR surface expression on primary mouse CD8 + T cells stably expressing CD28TM and proCAR-4 analyzed by c-Myc staining on flow cytometry. HER2 proCAR-4 was designed using the proMP C4.1 sequence without the final C-terminal leucine as a transmembrane domain (TMD), inserted as shown in . ( d ) IncuCyte killing assay over 24 hr of no CAR, CD28TM, and proCAR1-4 T cells on MC57-HER2 target cells at 1:1 effector to target ratio. Comparison of maximum killing for n = 6 independent experiments shown between CD28TM vs. ProCAR-4. Data points represent individual experiments, with mean ± SEM error bars plotted. ( e ) Tumor growth over time using the same experimental design in for No CAR (empty vector), CD28TM WT, and proCAR-4 T cell groups (n = 5–6 mice/group). Data points represent mean ± error bars showing SEM. Statistical analysis performed using a two-way ANOVA at day 10. ( f, g ) Linear correlation of proCAR oligomeric state vs. IFNγ, IL-2, TNFα, and GM-CSF cytokine production (normalized to CD28TM reference) from 24 hr co-culture with ( e ) MC57-HER2 and ( f ) antigen-negative MC57 tumor cells. Individual data points are colored, mean values in white box and error bars indicate SEM.

Article Snippet: Peptide, recombinant protein , Recombinant mouse TNFα (ELISA Std.) , BioLegend , Cat# 575209 , .

Techniques: SDS Page, Migration, Membrane, Sequencing, Expressing, Stable Transfection, Staining, Flow Cytometry, Comparison, Plasmid Preparation, Co-Culture Assay

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 1. Activation of Frizzled-1 by Wnt3a increases intracellular IP5. A, F9 clones stably expressing rat Fz1 were labeled with [3H]inositol for 48 h as described under “Experimental Procedures.” Cells were treated with Wnt3a (15 ng/ml) for the time periods indicated. Radiolabeled inositol phosphates were isolated from cell lysates and separated by strong anion HPLC. Repre- sentative chromatograms are shown. The retention times for IP3, IP4, IP5, and IP6 standards were determined and are displayed. B, a quantitative analysis of intracellular IP3, IP4, and IP5 in response to Wnt3a stimulation is shown. *, p 0.01; **, p 0.001, versus corresponding intracellular IPs measured at time 0. C and D, F9 clones stably transfected with EV, or expressing vector harboring either rat Fz1 or rat Fz2 were grown in 35-mm dishes and labeled with [3H]inositol for 48 h. Empty vector-transfected cells and F9 clones expressing Fz1 were stimulated by Wnt3a (15 ng/ml) for up to 60 min. Fz2-expressing clones were stimulated by Wnt5a (60 ng/ml) for up to 60 min. Cells were lysed at the indicated time periods and radiolabeled inositol phosphates were extracted and separated. C, radioactivity of [3H]IP5 extracted from samples was counted and a quantitative analysis of intracellular IP5 in response to Wnt stimulation is shown. *, p 0.01; **, p 0.001, versus the values obtained from empty vector-transfected cells stimulated with Wnt3a at time 0.

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Activation Assay, Clone Assay, Stable Transfection, Expressing, Labeling, Isolation, Transfection, Plasmid Preparation, Radioactivity

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 2. Gq mediates the increase in intracellular IP5 in response to Wnt3a. F9 clones stably expressing the Fz1 and Super8X TOPflash (as a lucif- erase-coupled gene reporter to measure Lef/Tcf-sensitive transcription) were either treated with siRNA for 60 h to knockdown Gq, Go, or G11 (A–C) or transiently transfected with constitutively activated G proteins (D). A, repre- sentative immunoblots (IB) of G-protein subunits in cells treated with siRNA to individually knockdown Gq, Go, or G11. Cells were treated for 60 h with siRNA. The cells were lysed and subjected to SDS-PAGE and immunoblotting. The blots of resolved proteins were stained for each subunit. -Actin was probed as a loading control. B, clones treated with siRNA were stimulated by Wnt3a for 6 h and Lef/Tcf-sensitive luciferase reporter gene activity was assayed. C, F9 clones stably expressing the Fz1 were metabolically labeled with[3H]inositolforatotal84h.Twenty-fourhafterthestartoftheincubation in the media containing [3H]inositol, cells were treated with siRNA reagents targeting Gq, Go, or G11 in the same media for 60 h. Cells were serum- starved for 8 h prior to stimulation for 15 min with Wnt3a (15 ng/ml). Labeled inositol polyphosphates were isolated and quantified. The IP5 levels were determined. Results are displayed as mean S.E., derived from at least three separate experiments. *, p 0.01; **, p 0.001; versus control siRNA-treated andWnt3a-stimulatedgroups;settingthevalueofcontrolsiRNA-treatedcells without stimulation as “1.” D, F9 clones were metabolically labeled with [3H]inositol for 48 h. Twenty-four hours after the start of the incubation in the mediacontaining[3H]inositol,clonesweretransientlytransfectedwithoneof following plasmids; expression vectors harboring Q209LGq (QLGq), or Q205LG (QLGo), or Q209LG11 (QLG11) (all with an EE-tag). Cellular [3H]IP3and[3H]IP5wereisolatedandcomparedforcellstransfectedwithEVor oneoftheCAmutantGproteinsubunits.ResultsaredisplayedasmeanS.E., derived from at least three separate experiments. *, p 0.01; **, p 0.001 for the difference between QL mutant-expressing and EV control cells. Repre- sentative immunoblots of EE-tagged QLGq, QLGo, and QLG11 expressed in these cells are shown. -Actin was probed as a loading control.

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Clone Assay, Stable Transfection, Expressing, Knockdown, Transfection, Western Blot, SDS Page, Staining, Control, Luciferase, Activity Assay, Metabolic Labelling, Labeling, Isolation, Derivative Assay, Incubation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 3. PLC1/PLC3 mediates Wnt3a-stimulated IP5 accumulation. A, RNA was isolated from F9 clones stably transfected with rat Fz1 and Super8X TOPflash without (upper panel) or with (lower panel) siRNA for 48 h. RT was employed followed by PCR amplification using primers specific for mRNAofeachindividualPLCisoform.RepresentativeresultsofRT-PCRampli- fications are displayed. Cyclophylin A mRNA was amplified as a control. B, Fz1-expressing cells were treated with siRNA for 48 h prior to the stimula- tion of Wnt3a (15 ng/ml) and luciferase reporter activity was assayed 6 h after the administration of Wnt3a. C, F9 clones stably expressing the Fz1 were metabolically labeled with [3H]inositol for 72 h. Twenty-four hours after the start of the incubation in the media containing [3H]inositol, cells were treated with either control siRNA or siRNA targeting both PLC1 and PLC3 in com- bination in the same media for 48 h. Cells were serum-starved for 8 h prior to stimulation with Wnt3a (15 ng/ml). Fifteen minutes after Wnt3a administra- tion, inositol polyphosphates were isolated from whole cell extracts, sepa- rated, and quantified as described. The results are displayed as mean S.E., derived from at least three separate experiments. **, p 0.001, versus Wnt3a- treated, control groups; #, p 0.01, versus Wnt3a-treated cells pretreated with siRNA to PLC1 or PLC3, individually.

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Isolation, Clone Assay, Stable Transfection, Transfection, Amplification, Control, Expressing, Luciferase, Activity Assay, Metabolic Labelling, Labeling, Incubation, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 4. Wnt3a-induced accumulation of intracellular IP5 is derived from the action of both IP3K and IPMK. A, schematic diagram of proposed pathways for IP5 synthesis in vivo. Activation of PLC hydrolyzes PI(4,5)P2 and generates diacylglycerol (DAG) and inositol 1,4,5-P3. IP3K and IPMK convert inositol (1,4,5)P3 to inositol1,3,4,5-P4andinositol1,4,5,6-P4,respectively.IPMKfurtherconvertsbothinositol1,3,4,5-P4andinositol 1,4,5,6-P4 to inositol 1,3,4,5,6-P5. Adriamycin inhibits IP3K, CGA inhibits IPMK, whereas ATA inhibits both IP3K and IPMK. B, inhibition of either IPMK or IP3K blocks Wnt3a-induced IP5 accumulation. Mouse F9 clones expressing Fz1 were metabolically labeled with [3H]inositol for 48 h. Clones were treated without or with ATA (2 M), CGA (40 M), or adriamycin (20 M) for 30–60 min prior to the addition of the Wnt3a (15 ng/ml). Fifteen min after Wnt3a stimulation, intracellular IP5 accumulation was measured. The results are displayed as mean S.E., derived from at least three separate experiments. *, p 0.001, versus correspondent control groups. C, suppression of IP3K and IPMK using siRNA reagents: analysis by immunoblotting (IB) (IP3K) and RT-PCR amplification (IMPK). -Actin was employed as a loading control for immunoblotting, whereas cyclophilin A was employed in the same set of samples amplified by RT-PCR. D, F9 clones expressing Fz1 were metabolically labeled with [3H]inositol for 72 h. Twenty-four hours after the start of the incubation in the media containing [3H]inositol, cells were treated with either control siRNA or siRNA reagents targeting either IPMK or IP3K-B, or siRNA targeting both IPMK and IP3K-B in combination. Cells were serum-starved for 8 h prior to stimulation for 15 min without or with Wnt3a (15 ng/ml). Intracellular accumulation of IP3 and IP5 were monitored by HPLC. The results are displayed as mean S.E., derived from at least three separate experiments. *, p 0.001, versus correspondent,controlsiRNA-treatedcells.#,p0.001,versuscellstreatedwiththesamesiRNAintheabsence of Wnt3a (Wnt3a). &, p 0.01, versus siRNA-treated cells in which either IPMK or IP3K-B alone were suppressed.

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Derivative Assay, In Vivo, Activation Assay, Inhibition, Clone Assay, Expressing, Metabolic Labelling, Labeling, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Incubation

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 5. Signaling in Wnt canonical pathway to -catenin, gene transcription, and PE formation. F9 clones expressing the Fz1 and Super8X TOPflash were stimulated by Wnt3a. Lef/Tcf-dependent transcription (B–E) and accumulationofcytoplasmic-catenin(A)weremeasured.TheresultsaredisplayedasmeanS.E.,obtainedfrom atleastthreeseparateexperiments.Endodermformation(F)wasanalyzedatday4afterWnt3atreatment.A,effects of pretreating cells with either ATA or adriamycin (as described in the legend to Fig. 4) on the accumulation of cytoplasmic -catenin in the absence versus the presence of Wnt3a. B, clones pre-treated with ATA, CGA, and adriaymycin were stimulated without or with Wnt3a and the Lef/Tcf-sensitive luciferase reporter activity deter- mined.**,p0.001,forthedifferencefromcellstreatedonlywithWnt3a(noinhibitor).C,clonesweretreatedwith siRNA for 48 h to knockdown either IPMK or IP3K-B, or both prior to Wnt3a stimulation. The Lef/Tcf-sensitive luciferase reporter activity was measured. **, p 0.001, versus cells treated with Wnt3a only, not treated with siRNAs targeting IP3K, IPMK, or both in tandem. #, p 0.01, versus cells treated with either “siRNA to IP3K-B” alone or “siRNA to IPMK” alone. D, Fz1-expressing cells were transiently transfected for 24 h with increasing amounts of plasmid harboring Myc-tagged IPMK as indicated. Clones were stimulated by Wnt3a and Lef/Tcf- sensitive luciferase transcriptional activity was measured. The expression of Myc-tagged IPMK in transfected clones was analyzed by immunoblotting (IB) with anti-Myc antibody (lower panel). *, p 0.01; **, p 0.001, for the difference from Wnt3a-treated control cells. E, Fz1-expressing cells were treated with siRNA for 24 h to knockdown IPMK. These knockdown cells were transiently transfected to express exogenous, Myc-tagged IPMK. Cells were stimulated without or with Wnt3a and the Lef/Tcf-sensitive luciferase reporter gene activity measured. *, p 0.01; **, p 0.001 versus Wnt3a-treated control cells. F, cells were treated with either control siRNA or siRNAs targeting IPMK as well as IP3K-B in combination for 48 h. Cells then were treated without or with Wnt3a (15 ng/ml) for 4 days and the formation of PE examined by immunostaining of the fixed cells with TROMA-1 antibody, a secondary fluorescently labeled antibody, in tandem with indirect immunofluoresence. PC, phase-contrast. IIF, indirect immunofluorescence. Bar, 20 m.

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Clone Assay, Expressing, Luciferase, Activity Assay, Knockdown, Transfection, Plasmid Preparation, Western Blot, Control, Immunostaining, Labeling, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 6. IP5 enhances CK2 activity in vitro. A, F9 clones expressing Fz1 were pre-treated with inhibitors to PLC (ET-18-OH), IPMK (CGA), or IPMK and IP3K (ATA) for 20 min prior to the stimulation by Wnt3a. Following

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Activity Assay, In Vitro, Clone Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 7. IP5 suppresses GSK3 activity in vitro. A, F9 clones expressing Fz1 were pre-treated with inhibitors to PLC (ET-18-OH), IPMK (CGA), or IPMK and IP3K (ATA) for 20 min prior to the stimulation by Wnt3a. GSK3 was

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Activity Assay, In Vitro, Clone Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Inositol Pentakisphosphate Mediates Wnt/β-Catenin Signaling

doi: 10.1074/jbc.m702106200

Figure Lengend Snippet: FIGURE 8. IP5: pivotal regulation of GSK3 and CK2 in Frizzled1/-cate- nin/Lef-Tcf signaling pathway. Wnt3a binds to Frizzled1 (FZ1), a member of the superfamily of G protein-coupled receptors, and thereby activates Gq, which in turn activates PLC. Activation of PLC leads to hydrolysis of phos- phatidylinositol 4,5-bisphosphate and generates IP3 and diacylglyceride (DAG). IP3 is quickly converted to IP5, the combined action of IPMK and IP3K, and does not appreciably bind to the IP3 receptor altering intracellular Ca2. Increase of intracellular IP5 accumulation stimulated (indirectly) the activa- tion of CK2 while inhibiting (indirectly) GSK3 activity. Wnt-stimulated -catenin stabilization and Lef/Tcf-sensitive transcriptional activation by Wnt3a are dependent upon the accumulation of IP5. KD, knock down by siRNA; IP3R, IP3 receptor.

Article Snippet: Recombinant mouse Wnt3a and Wnt5a were provided by R&D System (Minneapolis, MN).

Techniques: Activation Assay, Activity Assay, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: A mutation in NLRC4 induces overproduction of IL-1β through activation of caspase-1. (a) Flag-tagged wild-type, mutant NLRC4 expression vectors or empty vector (EV) were transfected into 293T cells. 2 d later, after the cell lysates were subjected to SDS-PAGE, Western blotting was performed with an anti-Flag or anti-actin antibody. Red triangle indicates NLRC4. The data in the figure are representative of three independent experiments. (b) Cell lysates from 293T cells transfected with empty vector (EV), flag-tagged wild-type, or mutant NLRC4 were subjected to Blue Native PAGE and the expression of the tagged proteins or actin was evaluated by Western blotting with an anti-Flag or actin antibody, respectively. The data in the figure are representative of three independent experiments. (c) 293T cells were transfected with a CASP1 expression vector together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the expression of procaspase-1, cleaved active caspase-1 (10 kD), and actin was evaluated by Western blotting. Red triangles indicate procaspase-1, cleaved active caspase-1, and actin. The data in the figure are representative of five independent experiments. (d) 293T cells were transfected with expression vectors for CASP1 and IL1B together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the concentration of IL-1β in the supernatant was evaluated by ELISA. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (e) Peripheral blood mononuclear cells were transfected with three concentrations of Prgl and cultured for 48 h. The concentrations of IL-1β in the supernatants were measured by ELISA. The data shown are means ± SD (**, P < 0.01). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Blue Native PAGE, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: BM-derived macrophages that overexpress mutant Nlrc4 secrete IL-1β. BM-derived macrophages (BMMC) infected with control retrovirus, retroviruses encoding wild-type, or mutant Nlrc4 were stimulated with 10 µg/ml LPS for 1 d in the absence (black) or presence (gray) of a caspase-1 inhibitor. As the control, unstimulated BMMCs were used (white). The concentrations of IL-1β and IL-6 in the supernatant were measured by ELISA. The data shown are means ± SD (*, P < 0.05; n = 5). The data shown in this figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Derivative Assay, Mutagenesis, Infection, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cytokine production in mu-Nlrc4 mice or mice reconstituted with mutant Nlrc4 transduced BM cells. (a) Splenocytes from wild-type or mu-Nlrc4 mice were stimulated with LPS for 24 h, and the concentration of IL-1β in each supernatant was evaluated by ELISA. The data shown are means ± SD (**, P < 0.01). N.D.: not detected. The data in the figure are representative of three independent experiments. (b) The concentrations of IL-1β, IL-17A, and G-CSF in the serum of wild-type (white) and mu-Nlrc4 (black) mice at the age of 8 wk were evaluated by ELISA. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of three independent experiments. (c) Intraperitoneal cells or splenocytes from C57BL/6 mice transplanted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus were evaluated for the number of Gr1 + CD11b + cells 2 mo after the transplantation. The data shown are means ± SD (*, P < 0.05; n = 5 in each group). The data in the figure are representative of three independent experiments. (d) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. Serum IL-1β, IL-17A, and G-CSF were evaluated by ELISA 6 wk after BM transplantation. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Control, Virus, Transplantation Assay, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cold exposure of mu-Nlrc4 mice increases autoinflammation. (a) The footpads of C57BL/6 (wild type; white) and mu-Nlrc4 (black) mice were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (b) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. The footpads were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (c) The entire bodies of control C57BL/6 and mu-Nlrc4 mice were exposed to 4°C for 1 min and kept for 3 min at room temperature, and then the serum concentrations of IL-1β were measured. The data are shown as means ± SD. N.D., not detected. (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (d) MC/9 cells infected with control retrovirus (white), retroviruses encoding wild-type (black), or mutant Nlrc4 (gray) were cultured at 32 or 37°C for 48 h. 293T cells were transfected with control vector (white), vectors encoding wild-type (black), or mutant NLRC4 (gray) and 48 h later, cells were washed and cultured at 32 or 37°C for 6 h. The levels of IL-1β in the supernatants were evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (*, P < 0.05). The data in the figure are representative of three independent experiments. (e) Peripheral mononuclear cells from an FCAS patient or a healthy control were cultured at 32°C for 48 h in the presence (black) or absence (white) of a caspase inhibitor or cultured at 37°C for 48 h. The level of IL-1β in the supernatant was evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (**, P < 0.01). The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Irradiation, Infection, Control, Virus, Mutagenesis, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: The accumulation of neutrophils in mu-Nlrc4 mice depends on IL-1β and IL-17A. (a) Spleen cells from C57BL/6 or mu-Nlrc4 mice at the age of 12 wk were stimulated with 25 ng/ml PMA and 1 µg/ml ionomycin in the presence of 2 µM monensin for 5 h and then stained with anti–TCR-β, anti–TCR-γδ, anti-B220, anti-CD11c, anti-NK1.1, anti-Gr1, and anti-CD11b antibodies. The cells were fixed and stained with anti–IL-17A antibody. The expression of IL-17A was analyzed by flow cytometry. The number in each rectangle indicates the percentage of IL-17A–positive cells in the lineage-negative fraction. The data in the figure are representative of five independent experiments. (b) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti-CD4, anti-CD8, anti-Thy1.2, anti-Gr1, or anti–IL-1β antibody five times at 3-d intervals. The serum IL-17A levels 2 d after final antibody treatment was measured by ELISA. Control sera from C57BL/6 (wild type) mice was used. The data shown are means ± SD (**, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments. (c) mu-Nlrc4 mice at the age of 12 wk were injected twice with control rat IgG, anti-CD4, anti-CD8, or anti-Thy1.2 at 3 d intervals. The spleen cells from mice 1 d after the final injection were stained with anti-CD4 and anti–TCR-β (for anti-CD8–treated mice), anti-CD8 and anti–TCR-β (for anti-CD4–treated mice), or anti-CD4, anti-CD8, and anti–TCR-β antibodies (for anti-Thy1.2–treated mice). The data in the figure are representative of two independent experiments. (d) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti–IL-1β, anti–IL-17A, or anti–IL-1β, and anti–IL-17A antibody five times at 3-d intervals. The number of Gr1 + CD11b + cells in the spleen 2 d after the final antibody treatment was evaluated by flow cytometry. The data shown are means ± SD (*, P < 0.05; **, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Staining, Expressing, Flow Cytometry, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases

doi: 10.3389/fimmu.2022.1054472

Figure Lengend Snippet: Generation of nucleus-deliverable ndSTAT1-TMD and verification of its intra-nuclear transduction kinetics without cellular cytotoxicity. (A) Protein structure of STAT1-TMD without Hph-1-PTD, ndSTAT-TMD, and ndSTAT1-TMD (V426D, T427D) with mutated valine 426, and threonine 427. PTD, protein transduction domain; nd, nucleus-transducible; TMD, transcription modulation domain; DBD, DNA binding domain; ND, N-terminal domain; LD, linker domain; SH2, SCR2 homology domain; TAD, transactivation domain. (B) The identity of the purified STAT1-TMD, ndSTAT1-TMD, and ndSTAT1-TMD (V426D, T427D) was confirmed by western blot (left panel) or SDS-PAGE (right panel). (C, D) Dose-dependent and time-dependent intra-nuclear delivery of ndSTAT1-TMD (0.5-2 μM) or STAT1-TMD (2 μM) into mouse splenocytes. (E) Intracellular stability of ndSTAT1-TMD after transduction into mouse splenocytes. (F) Intracellular localization of ndSTAT1-TMD in cultured mouse splenocytes. Representative immunofluorescence 63X (left panel) and 189X (right panel) images using a confocal microscope. Scale bar = 10 μm. (G) Evaluation of cellular cytotoxicity of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) in mouse splenocytes analyzed by CCK-8 assay. (H, I) The level of the induced expression of CD25, CD69, or IL-2 secretion in CD4 + T cells activated by anti-CD3ϵ antibody and anti-CD28 antibody in the presence of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) was analyzed by flow cytometry and ELISA. The graphs are represented as mean ± SEM (n=3), and the statistical analysis was examined using Student’s t-test. ns, not significant.

Article Snippet: The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc, USA.

Techniques: Transduction, Binding Assay, Purification, Western Blot, SDS Page, Cell Culture, Immunofluorescence, Microscopy, CCK-8 Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases

doi: 10.3389/fimmu.2022.1054472

Figure Lengend Snippet: ndSTAT1-TMD inhibits the expression of STAT1-target genes at the transcription level. (A) HEK293T cells were co-transfected with the plasmids containing the luciferase reporter gene, whose expression is driven by the IL-17A promoter, and the wild-type STAT1 gene. The luciferase activity of cells was measured 24 hr after the treatment with ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) by a luminometer. Data are represented as mean ± SEM (n=5), and statistical significance was indicated when the luciferase activity was lower than the positive control group (pCMV-STAT1-only). The statistical analysis was examined using Student’s t-test. ****p<0.0001. (B, C) GSEA-based enrichment plots of the hallmark of IFN-γ response gene set in ndSTAT1-TMD-treated Th1 cells or of the gene set upregulated in STAT3 knockout CD4 + T cells compared to wild-type CD4 + T cells in ndSTAT1-TMD-treated Th17 cells. (D, E) Heatmap of Th1 or Th17 cell signature genes and naïve T cell-specific genes in Th1 or Th17 cells treated with or without ndSTAT1-TMD. Each column means results from one experimental well Z-score was calculated from log2(TPM+1).

Article Snippet: The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc, USA.

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Positive Control, Knock-Out

Journal: Journal of cellular biochemistry

Article Title: AHNAK2 Participates in the Stress-Induced Nonclassical FGF1 Secretion Pathway.

doi: 10.1002/jcb.25047

Figure Lengend Snippet: Fig. 1. Mass spectrometric analysis of FGF1:HA immunoprecipitated proteins suggests AHNAK2 as a candidate binding protein at 42°C. (A) Partial sequence from human AHNAK2 showing high (<95% confidence, bold face font) and medium confidence (>90% confidence) peptides that were consistent with MSMS-TOF-derived protein sequence. (B) MSMS-TOF collision-induced decay protein sequence data supporting identification of the high confidence AHNAK2 peptide. (C) MSMS-TOF spectrum corresponding to the data in (B).

Article Snippet: Genetic constructs pGFP-V-RS expressing shRNAs that suppress the expression of mouse AHNAK2 were obtained from Origene (Rockville, MD).

Techniques: Immunoprecipitation, Binding Assay, Sequencing, Derivative Assay

Journal: Journal of cellular biochemistry

Article Title: AHNAK2 Participates in the Stress-Induced Nonclassical FGF1 Secretion Pathway.

doi: 10.1002/jcb.25047

Figure Lengend Snippet: Fig. 2. Heat shock enhances the association between FGF1 and C-terminus (ct) of AHNAK2. NIH 3T3 cells were adenovirally co-transduced with FGF1:HA and ctAHNAK2:V5 and incubated in serum-free medium at 37°C or 42°C for 90 min. Cell lysates were prepared. FGF1 was precipitated from cell lysates by using anti-HA antibodies (A) and ctAHNAK2 by using anti-V5 antibodies (B). Immunoprecipitates were resolved by SDS PAGE and immunoblotted by using anti-HA and anti-V5 antibodies.

Article Snippet: Genetic constructs pGFP-V-RS expressing shRNAs that suppress the expression of mouse AHNAK2 were obtained from Origene (Rockville, MD).

Techniques: Transduction, Incubation, SDS Page

Journal: Journal of cellular biochemistry

Article Title: AHNAK2 Participates in the Stress-Induced Nonclassical FGF1 Secretion Pathway.

doi: 10.1002/jcb.25047

Figure Lengend Snippet: Fig. 3. Heat shock induces a peripheral localization of FGF1 and ctAHNAK2. NIH 3T3 were adenovirally transduced with FGF1:HA and ctAHNAK2:V5 and plated on glass coverslips. Adherent co-transduced cells were incubated in serum-free medium at 37°C or stressed at 42°C for 90 min and then formalin fixed. Cells were co-stained with FITC- labeled anti-HA antibodies (green), CY3-labeled anti-V5 antibodies (red), DAPI (blue), and studied via a confocal microscope. Scale bar indicates 8 mm. Arrows indicate AHNAK2 and FGF1 colocalization at the periphery of a heat shocked cell.

Article Snippet: Genetic constructs pGFP-V-RS expressing shRNAs that suppress the expression of mouse AHNAK2 were obtained from Origene (Rockville, MD).

Techniques: Transduction, Incubation, Staining, Labeling, Microscopy

Journal: Journal of cellular biochemistry

Article Title: AHNAK2 Participates in the Stress-Induced Nonclassical FGF1 Secretion Pathway.

doi: 10.1002/jcb.25047

Figure Lengend Snippet: Fig. 4. Heat shock induces the association of FGF1 and ctAHNAK2 with the cytoskeleton. (A) NIH 3T3 cells transfected with FGF1:HA were subjected to a 90 min incubation at 42°C. FGF1 was detected by using FITC-labeled anti-HA antibodies (green) and F actin by using CY3-labeled phalloidin (red). Cells were studied via a confocal microscope. Arrow indicates FGF1 and F-actin colocalization at the cell periphery. Scale bar–32 mm. (B, C) NIH 3T3 cells transfected with FGF1:HA were incubated for 90 min at 37°C or 42°C. Cytosolic and cytoskeletal fractions were prepared by utilizing the Qproteome kit, resolved by SDS PAGE and immunoblotted with vimentin antibodies (cytoskeleton marker) (B) or FGF1 antibodies (C). (D) NIH 3T3 cells adenovirally transduced with ctAHNAK2:V5 were treated and fractionated similar to (B), ctAHNAK2 was detected by using anti-V5 antibodies. E. Co-localization of FGF1, AHNAK2 and F-actin at the periphery of a stressed cell. NIH 3T3 cells were co-transduced with FGF1:HA and ctAHNAK2:V5, and heat shocked. Fixed cells were immunofluorescently stained with FITC-labeled anti-HA antibodies (green), CY3-labeled anti-V5 antibodies (red) and Alexa 633-labeled phalloidin (blue), and studied via a confocal microscope. Scale bar–2 mm.

Article Snippet: Genetic constructs pGFP-V-RS expressing shRNAs that suppress the expression of mouse AHNAK2 were obtained from Origene (Rockville, MD).

Techniques: Transfection, Incubation, Labeling, Microscopy, SDS Page, Marker, Transduction, Staining

Journal: Journal of cellular biochemistry

Article Title: AHNAK2 Participates in the Stress-Induced Nonclassical FGF1 Secretion Pathway.

doi: 10.1002/jcb.25047

Figure Lengend Snippet: Fig. 5. shRNA knockdown of AHNAK2 expression inhibits the stress-induced FGF1 export. (A) NIH 3T3 cells were stably transfected with AHNAK2 shRNAs, scrambled shRNA or empty vector. AHNAK2 mRNA levels in transfected cells were assessed by RT PCR. Two of three shRNA constructs suppressed AHNAK2 expression. (B) AHNAK2 shRNA 1 and 2, and scrambled shRNA transfectant cells were adenovirally transduced with FGF1:HA and incubated for 90 min at 37°C or 42°C. FGF1 was isolated from the cell culture media by heparin chromatography. The levels of FGF1 in cell lysates and media were determined by SDS PAGE and immunoblotting using anti-FGF1 antibodies. (C, D) Quantification of the effect of shRNA 2 transfection on FGF1 release in a separate experiment. Immunoblotting (C) and photometric quantification (D) results are presented. FGF1 release from scrambled shRNA and AHNAK2 shRNA transfectants was normalized to FGF1 content in cell lysates at 37°C. The normalized heat shock-induced release from scrambled shRNA transfectants is presented as 100%. The data are representative of three independent shRNA experiments, which gave consistent results.

Article Snippet: Genetic constructs pGFP-V-RS expressing shRNAs that suppress the expression of mouse AHNAK2 were obtained from Origene (Rockville, MD).

Techniques: shRNA, Knockdown, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Construct, Transduction, Incubation, Isolation, Cell Culture, Chromatography, SDS Page, Western Blot

Journal: Journal of cellular biochemistry

Article Title: AHNAK2 Participates in the Stress-Induced Nonclassical FGF1 Secretion Pathway.

doi: 10.1002/jcb.25047

Figure Lengend Snippet: Fig. 6. (A) Stress-independent FGF1 export induced by the inhibition of Notch signaling does not depend on AHNAK2. NIH 3T3 cells stably transfected with scrambled shRNA or AHNAK2 shRNA (construct 2) were adenovirally co-transduced with FGF1:HA and dnCBF1. Cells were incubated for 90 min at 37°C or 42°C. FGF1 was isolated from the cell culture media by heparin chromatography. The levels of FGF1 in cell lysates and media were determined by SDS PAGE and immunoblotting using anti-FGF1 antibodies. dnCBF1 transduced cells released FGF1 at 37°C. AHNAK2 knockdown in dnCBF1 transduced cells did not affect the release of FGF1 at 37°C but prevented its enhancement by heat shock. (B) Spontaneous FGF2 export is insensitive to AHNAK2 depletion. NIH 3T3 cells stably transfected with scrambled shRNA or AHNAK2 shRNA (construct 2) were co-transduced with FGF2:HA and incubated for 90 min at 37°C or 42°C. FGF2:HA was isolated from the cell culture media by heparin chromatography. The levels of FGF2:HA in cell lysates and media were determined by SDS PAGE and immunoblotting using anti-HA antibodies. The data presented in A and B are each representative of three independent shRNA experiments, which gave consistent results.

Article Snippet: Genetic constructs pGFP-V-RS expressing shRNAs that suppress the expression of mouse AHNAK2 were obtained from Origene (Rockville, MD).

Techniques: Inhibition, Stable Transfection, Transfection, shRNA, Construct, Transduction, Incubation, Isolation, Cell Culture, Chromatography, SDS Page, Western Blot, Knockdown

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 1. Existence of Chk2-PP2A complex. (A) Immunostaining to locate sites of association between the two enzymes. A2780 cells were treated with 3 μM cisplatin for 1 h and double stained with anti-p-Chk2 antibodies (left, green) or anti-PP2A antibodies (middle, red) 16 h later. Colocalization of the two images is seen where the signal appears as yellow spots in the right panel. (B) Coimmunoprecipitation of PP2A with Chk2 and with HA-Chk2. Immunoprecipitations from cisplatin-treated A2780 cell extracts were performed with antibodies of anti-Chk2 (Chk2) or anti-normal mouse serum (NMS) (a). HA-Chk2 immunoprecipitates were obtained with anti-HA antibody from cells transfected with wild-type plasmid of HA-hChk2 pEFBOS and treated with cisplatin (b). Immune complexes were subjected to immunoblot analyses with anti-p-Chk2(Thr68), anti-Chk2, anti-PP2A-A, anti-PP2A-B and anti-PP2A-C antibodies.

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: Immunostaining, Staining, Transfection, Plasmid Preparation, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 2. (A) Cisplatin-induced Chk2 phosphorylation at Thr-68. A2780 ovarian cancer cells were treated with cisplatin (CDDP) at the IC50 dose (3 μM) for 1 h. The drug was removed by washing cells with PBS. Cells were then incubated in fresh drug-free media for the indicated hours (0, 3, 6, 12, 24 and 48) until harvest. Proteins were extracted and separated on SDS-PAGE gels. Blots were probed with antibodies of anti-p-Chk2 or anti- Chk2, or anti-ß-actin as a control. (B) Quantification of p-Chk2 expression was determined using IPLab-Gel software. (C) siRNA-mediated PP2A silencing affects the p-Chk2 level in A2780 cells. Cells were transfected with siRNA to the catalytic subunit of PP2A for 24 h and treated with 3 μM cisplatin for 1 h. The extent of phosphorylated Chk2 and p53, and the protein level of Chk2, PP2A and ß-actin were estimated using antibodies against respective p-Chk2(Thr68), p-p53(Ser15), Chk2, PP2A and ß-actin.

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: Phospho-proteomics, Incubation, SDS Page, Control, Expressing, Software, Transfection

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 3. (A) Inhibition of PP2A activity by okadaic acid enhances Chk2 phosphorylation. A2780 cells were pretreated with concentrations of OA (0, 5, 10 and 20 nM) for 30 min and then treated with 3 μM cisplatin for 1 h under continuous exposure to OA. Cells were washed to remove cisplatin and re-incubated for 24 h in a replacement of fresh media containing OA. Proteins were prepared, separated and probed with antibodies of anti-p-Chk2, anti-Chk2, or anti-ß-actin. ß-actin served as a control. (B) Inhibitory effects of okadaic acid on protein phosphatase activity by protein phosphatase activity assay. Cells were pretreated with okadaic acid and then treated with cisplatin as described above. The protein phosphatase activity assay was performed following the manufacturer's instructions. Results are expressed as the means ± SD (n=3).

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Incubation, Control, Phosphatase Assay

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 4. Direct dephosphorylation of p-Chk2 by PP2A in vitro. Chk2 was phosphorylated by cisplatin treatment to the A2780 cells. Forty μg of whole cell lysates (A), Chk2-immunoprecipitated protein (B), or HA-Chk2 immunoprecipitated protein (C) was incubated with 0.05 or 0.5 units of PP2A enzyme in the presence or absence of the phosphatase inhibitor OA (B and C), which was used to verify the specificity of the PP2A effect.

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: De-Phosphorylation Assay, In Vitro, Immunoprecipitation, Incubation

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls ploidy in Y235T cells. A The presented bar graph illustrates the GJB3 mRNA amounts across various human tissues, with detailed information available in the Materials and Methods section. Urothelial cells (UC#1 and UC#2) were isolated from ureters from two separate patients who underwent nephrectomy at Ulm University. The mRNA levels were normalized to GAPDH . n = 3 independent experiments were performed. Error bars represent mean ± SEM. B The representative pictures display the HE, GJB3 and IgG staining in human ureter tissues (U#1 and U#2, respectively). C The representative Western blot result indicates GJB3 protein levels in Y235T cells with shGJB3. α-tubulin is used as a loading control. n = 3 independent experiments were performed. D The bar graphs depict the effectiveness of GJB3 knockdowns at the mRNA level in Y235T cells, with the measurements reference to the GAPDH mRNA level. n = 3 independent experiments were performed. Error bars represent mean ± SEM. E Representative pictures showing metaphase spreads of Y235T cells with shControl and shGJB3#2. Chromosomes are visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Control cells showing 46 chromosomes in most metaphase spreads. Exemplary pictures demonstrating the induction of aneuploidy in Y235T cells subsequent to GJB3 knockdown. The images show a metaphase spread of Y235T-shGJB3#2 cells with 51 chromosomes. F Chromosomes numbers of metaphase spreads from Y235T cells that were knockdown GJB3. n = numbers of (Each counting is indicated within the graph). Results are pooled from three independent sets of experiments. Mean ± SEM values are shown in the dot plot, and significance was determined by using Fisher’s exact test. G Representative pictures showing micronuclei of Y235T cells with shGJB3#1. Cell nuclei are stained with DAPI, and phalloidin Alexa Fluor 488 was used for F-actin visualization. White arrows indicate micronuclei. H Quantitation of cells with micronuclei upon knockdown of GJB3. Results from n = 3 separate series of experiments. The bar graph displays the mean ± SEM values, and the two-tailed Student's t-test was used to assess the significance. I Immunofluorescence results indicate the multinucleation of Y235T shGJB3#1 cell. Cell nuclei is visualized by DAPI, and F-actin is visualized by Alexa Fluor 488. J Quantitation of cells with multinucleation with knockdown of GJB3. Results from n = 3 independent sets of experiments. Mean ± SEM values are shown in the bar graph, and the significance was determined by two-tailed Student’s t -test. K Figures depict of mitotic abnormalities in metaphase and anaphase. DAPI (blue) indicates chromosomes, Cy5 (red) indicates α-tubulin, and Alexa Fluor 488 (green) labeling illustrates γ-tubulin. White arrows are used to indicate chromatid mislocation or multipolar centrosomes. L – M Quantitative evaluation of mitotic abnormalities. Results from n = 3 distinct experiments. The bar graph displays mean ± SEM data, and a two-tailed Student’s t -test was used to assess significance. Scale bars: 200 μm ( B main panels) 50 μm ( B insets) 20 μm ( E , G , I ) and 2 μm ( K ). Images are shot at total magnification of 100x ( B main panels), 630x ( B insets, E , G , I , K )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Isolation, Staining, Western Blot, Control, Knockdown, Quantitation Assay, Two Tailed Test, Immunofluorescence, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls spindle orientation and microtubule dynamics. A Exemplary pictures illustrating the disorientation of Y235T-shGJB3#1 cells. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. B Quantitative assessment of the spindle pole displacement factor (SPDF) in Y235T cells. n = 256 (Y235T-shScr), 253 (Y235T-shGJB3#1), 263 (Y235T-shGJB3#2), C , Representative images showing reorientation of UMUC3 cells with ectopic GJB3. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. D Quantitative assessment of the spindle pole displacement factor (SPDF) in UMUC3 cells. n = 155 (UMUC3-EV), and 140 (UMUC3-GJB3). Experiments present combined data from three separate sets of independent experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. To boost the proportion of prometaphase cells, cells were treated to dimerthylenastron for four hours prior to labeling. E Examples of images demonstrating microtubule growth in Y235T cells expressing shGJB3#2. F Rates of mitotic microtubule plus-end assembly in Y235T-shGJB3 cells. n = 60 cells are pooled from three independent sets of experiments. G Example of images demonstrating growth of microtubules in UMUC3-GJB3 cells. H Rates of mitotic microtubule plus-end assembly in UMUC3-GJB3 cells. n = 60 cells are combined from three separate sets of experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. GJB3 interacts with α-tubulin. The deletion of GJB3 in UROtsa cells using the CRISPR-cas9 method was detailed in the main article. I Western blot displaying GJB3 protein levels in UROtsa cells with a control guide RNAs directed against green flourescent protein or two distinct gRNAs targeting GJB3 (gGJB3#1 and GJB3#2). α-tubulin serves as loading control. n = 3 separate experiments were conducted. J Exemplary pictures displaying GJB3 and α-tubulin colocalization in UROtsa cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. K Quantitation of GJB3 and α-tubulin colocalization in UROtsa cells by Pearson’s correlation coefficient. n = 49 (UROtsa-gControl), 58 (UROtsa-gGJB3#1), 53(UROtsa-gGJB3#2) are pooled from three to four independent experiments. L GJB3 protein level in UMUC3 cells with ectopic GJB3 was detected by Western blot. α-tubulin is used as a loading control. n = 3 independent experiments were performed. M Representative images displaying the colocalization of GJB3 and α-tubulin in UMUC3 cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. N Quantitation of GJB3 and α-tubulin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 105 (UMUC3-EV), and 64 (UMUC3-GJB3) are pooled from three to four independent experiments. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t-test ( M , N ). O – P GJB3 bundle microtubule (MT) filament level was detected by Western blot. 5 × 10 11 MT/ml and 5–10 μm in length MTs were incubated with increasing concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . ( O ), Flag-GJB3, indicated by red arrowheads and ( P ), microtubules, indicated by red arrows, are visualized by specific antibodies. n = 3 independent experiments were performed. Scale bars: 5 μm ( A , C) and 1 μm ( E , G) and 2 μm ( J , M ). Images were captured at total magnification of 630x

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Two Tailed Test, Labeling, Expressing, CRISPR, Western Blot, Control, Quantitation Assay, Incubation, SDS Page, Centrifugation

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 as tumor suppressors in bladder cancer. A–B ( A ) RT-qPCR was used to measure the GJB3 mRNA expression , and ( B ) Western blot was employed to ascertain protein amounts. The relative mRNA expression was quantified with respect to GAPDH . The qPCR shows results of n = 4 technical repeats. Error bars represent Mean ± SEM. α-tubulin serves as loading control in Western blot. n = 3 independent experiments were performed. C Comparison of GJB3 mRNA levels in the CNUH (GSE13507) cohort of NMIBC ( n = 103) and MIBC ( n = 62) human bladder tumors. Statistical differences were defined by two-way Fisher’s ANOVA test * = p ≤ 0.05. D The IHC images represent the GJB3 staining (red arrows) in human normal bladder and bladder cancer tissues. E Quantitation of GJB3-IHC staining scores in human normal bladder and bladder cancer groups. F The IHC images display Gjb3 staining (red arrows) in mouse normal bladder and bladder cancer tissues during BBN-induced BC progression. G Quantitation of Gjb3-IHC staining scores in normal bladder and bladder cancer tissues during the BBN-induced BC progression in mice. The expression of Gjb3 gradually diminished after the BBN treatment, in contrast to the control mice (black dots), which were given water (orange dots). The bar graph displays mean ± SEM data, and a two-tailed Student's t-test was used to assess significance. Scale bars: 200 μm ( D and F , main panels) and 50 μm ( D and F insets). Images were captured at total magnification of 100 × ( D and F main panels), and 630 × ( D and F insets)

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Comparison, Staining, Quantitation Assay, Immunohistochemistry, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 inhibits cells invasion and migration. A – B Western blots displaying the GJB3 protein quantity assessments in RT4 and in UMUC3 cells with experimental modifications. The loading control was provided by α-tubulin levels. The Western blots were repeated for three times. C Cell migratory capacity in RT4 cell line with shGJB3#1 was detected by Wound healing/scratch. The exemplary imaged were captured at 24 and 120 h. D Normalized cell free area was used to quantify the impact of GJB3 knockdown on RT4 cells by Wound healing assay. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. E Cell migratory capacity in UMUC3 cell line with ectopic GJB3 was detected by Wound healing/scratch. The representative pictures captured at 12 and 24 h in case of UMUC3 cells. F Quantitation of normalized cell free area of UMUC3 cells with ectopic GJB3 performed by Wound healing assay n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. G The invasion capacity of RT4 cell line with shGJB3#1 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 144 h post-seeding. H Quantitation of invasive capacity of RT4 cells expressing the indicated shRNAs targeting GJB3. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. I The invasion capacity of UMUC3 cell line with ectopic of GJB3 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 48 h post-seeding. J, Quantitation of invasive capacity of UMUC3 cells with ectopic GJB3 expression. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. K Representative images of hematoxylin/eosin stainings showing the invasion capacity of RT4 cell line with shGJB3#2 by porcine bladder ex vivo organ culture method (the invasive capacity of BC cells in the ex vivo organ culture model was quantified as shown in Fig. S4). The cells were seeded on the surface of the de-epithelized porcine bladder for 21 days. L Quantitation graphs displaying the impact of GJB3 alteration on the invasive capacity of RT4 cells in ex vivo organ culture approach. n = 4 (RT4-shScr), n = 3 (RT4-shGJB3#1), n = 3 (RT4-shGJB3#2) M Representative images of hematoxylin/eosin stainings showing the invasion capacity of UMUC3 cell line with ectopic GJB3 by porcine bladder ex vivo organ culture approach. The cells were seeded on the surface of the de-epithelized porcine bladder for or 14 days. Insets: enlarged images of the areas shown by black boxes. Black arrows indicate the cells that have spread the farthest from the surface. n = 3 (UMUC3-EV), n = 4 (UMUC3-GJB3) independent experiments were performed. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t -test. Scale bars: 200 μm ( C , E , G , I , K main panels, M main panels) and 100 μm ( K insets, M insets). Images were captured at total magnification of 50 × ( C , E ), 100 × ( G , I , K main panels, M main panels), and 200 × ( K insets, M insets). O - V Morphological changes and actin-enriched protrusions in UMUC3, and RT4 cells with altered GJB3 expression. Cells with actin-enriched protrusions are marked with white arrows. O - P Brightfield microscopy images depict cells exhibiting a transition towards a round morphology ( O ) of UMUC3 cells with ectopic GJB3 expression. P RT4 cells with GJB3 knockdown demonstrate an elongated shape. Magnifications indicate 100 × or 400 × , respectively. The scale bars refer to 100 μm (left), and 50 µm (right). Q - T Quantification of round or elongated morphology on fixed cells. A cell with elongated or round morphology is identified by the ratio of longest and shortest diameter of the cell from images captured randomly at 630 × magnification using a Zeiss TCS SP5 confocal microscope. Scale bars: 20 μm. The ratio is calculated as the longest diameter of the cell dividing by the shortest diameter of the cell. The ratio is calculated as longest diameter dividing by shortest diameter. A cells with Ratio ≤ 2 is identified as round morphology, while ratio > 2 is elongated morphology. Q , R Immunofluorescence staining photos illustrate the round morphology shifting of ( Q ) UMUC3 cells with GJB3 overexpression compared to cells transfected with empty vector (EV). R RT4 cells with GJB3 knockdown display a transition towards an elongated morphology compared to cells transfected with shScramble (shScr). Cell nuclei are stained with DAPI, and F-actin is labeled with Phalloidin-AF488. S , T The bar graphs reveals percentages of cells with different morphology in total in each group, shown above in Q and R . The percentage was calculated as number of cells with elongated (or rounded) morphology divide cell numbers in total. The percentage values in different groups are marked above or in the bars. Black bars indicate percentages of cells with elongated morphology, and gray bars indicate the percentage of cells with rounded morphology. The statistical significance is calculated by using chi-square statistic. U , V The graphs show the fraction of cells with actin-enriched protrusions in response to GJB3 alterations ( U ) in RT4 or ( V ) in UMUC3 cells. For each picture, the percentage of cells with actin-enriched protrusions is calculated by the number of the cells with actin-enriched protrusions divided by total number of cells. (n) indicates the number of pictures taken in the group

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Migration, Western Blot, Control, Knockdown, Wound Healing Assay, Two Tailed Test, Quantitation Assay, Staining, Expressing, Ex Vivo, Organ Culture, Microscopy, Immunofluorescence, Over Expression, Transfection, Plasmid Preparation, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 interactics with F-actin and influences invadopodia formation via actin dynamics. A Representative images demonstrating invadopodia formation of RT4 cells with shGJB3#2. B Quantitation of the invadopodia number was performed in RT4 cells. n = 959 (RT4-shScr), n = 718 (RT4-shGJB3#1), n = 481 (RT4-shGJB3#2). Cells are pooled from three independent sets of experiments. C, Representative pictures showing invadopodia formation of UMUC3 cells upon ectopic GJB3 expression. D Quantitation of the invadopodia number was performed in UMUC3 cells. n = 110 (UMUC3-EV), n = 137 (UMUC3-GJB3). Cells are pooled from three independent sets of experiments. F-actin is visualized by Alexa Fluor 488-phalloidin and Cortactin is visualized by Cy5, respectively. Yellow spots displaying cortactin and F-actin colocalization identify the invadopodia structures. The regions indicated by white boxes are magnified in the insets. The invadopodia are marked with white arrows. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. E Representative pictures indicate the gelatin degradation by RT4 cells upon GJB3 knockdown. F Gelatin degradation capacity of the cells was quantified by measuring the degradation area per RT4 cell. n = 2809 (RT4-shScr), n = 2447 (RT4-shGJB3#1), n = 3544 (RT4-shGJB3#2). G Representative pictures indicate the gelatin degradation by UMUC3-GJB3 cells. H Gelatin degradation capacity of the cells was quantified by measuring the degradation area per UMUC3 cell. n = 1048 (UMUC3-EV), and n = 1246 (UMUC3-GJB3) are pooled from three to four independent experiments. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. I The LifeAct–Ruby signal recovery duration in UMUC3 cells with or without GJB3 is depicted in representative images after LifeAct–Ruby signal bleaching. The white arrows indicate the areas of bleaching. J , The quantitation of bleaching recovery experiments with UMUC3 cells. n = 29 (UMUC3-EV), n = 31 (UMUC3-GJB3). Three different groups of separate experiments' cells are combined. The graphs' data points correspond to the mean ± SEM. P values were calculated using the two-tailed Student's t -test at t = 49 s. K Exemplary pictures displaying the colocalization of GJB3 with F-actin in control UROtsa cells. Insets: enlarged image of the areas shown by white box. L , Quantitation of GJB3 and F-actin colocalization in UROtsa by Pearson’s correlation coefficient. n = 161 (UROtsa-gControl), n = 206 (UROtsa-gGJB3#1), n = 276 (UROtsa-gGJB3#2). M Exemplary pictures displaying the GJB3/F-actin colocalization in UMUC3-GJB3 cells. Insets: enlarged image of the areas shown by white box. N Quantitation of GJB3 and F-actin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 672 (UMUC3-EV), and n = 831 (UMUC3-GJB3) are combined from 3 separate experiments. The two-tailed Student's t-test was used to establish significance, and the bar graph displays mean ± SEM results. Alexa Fluor 647 illustrates the F-actin, and Alexa Fluor 488 illustrates GJB3. Yellow highlights denote GJB3 and F-actin overlap. Insets: enlarged images of the colocalized areas shown by white boxes. O , P GJB3 binds bundle actin filaments in a dose-dependent manner by Western blot. Actin (2.5 mg/ml) concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . Red arrowheads indicate the GJB3, and the red arrow indicates actin filaments visualized by western blot with specific antibodies. n = 3 independent experiments were performed. Scale bars: 10 μm ( A , C , E , G , K and M main panels), 1 μm ( A , C insets), 2 μm ( E , G , K and M insets) and 1 μm ( I ). Images were captured at total magnification of 630 × ( A , C , I , K , M ) and 400 × ( E , G )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitation Assay, Expressing, Two Tailed Test, Knockdown, Control, Western Blot, SDS Page, Centrifugation

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Identification of the heat shock proteins HSP70 and HSP90 as putative ChAT protein-interactors by proximity-dependent biotin identification (BioID). (A) Optimization of BioID in HEK293 cells expressing wild-type or P17A/P19A-ChAT fused to the HA-tagged promiscuous biotin ligase BirA-R118G (BirA * ). Control cells were transfected with empty vector or plasmids encoding either untagged ChAT or BirA * . Cells were treated for 24 h with either 50 μM biotin to facilitate proximity-dependent biotinylation of ChAT-interacting cellular proteins or with vehicle-control (water). Biotinylated proteins were isolated from cell lysates by streptavidin pull-downs (PD: Strep) and immunoblotted as indicated ( n = 2). (B) Identification of HSP70 and HSP90 as ChAT proximally-interacting proteins. Streptavidin PD samples prepared from biotin-treated HEK293 cells expressing wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * fusion proteins were resolved and visualized on a silver-stained SDS-PAGE gels. Two proteins (~70 and ~90 kDa) that were enriched in samples expressing P17A/P19A-ChAT-BirA * were identified by MALDI-TOF-MS or LC-ESI-MS/MS as HSP70 and HSP90, respectively. Control cells were transfected to express untagged wild-type ChAT ( n = 1). (C) Confirmation of endogenous HSP70 and HSP90 as putative ChAT-interacting proteins by immunoblotting of streptavidin PD samples prepared from biotin-treated HEK293 cells expressing HA-tagged wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * . Control cells were transfected with empty vector or vector encoding untagged ChAT ( n = 4).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Staining, SDS Page, Tandem Mass Spectroscopy, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSP90 ATPase activity specifically reduces steady-state levels of mutant ChAT protein. Immunoblots from SN56 cells expressing wild-type (A) , P17A/P19A- (B) , V18M- (C) , or A513T-ChAT (D) that were treated for 24 h with 0.5–2 μM with 17-AAG, an inhibitor of HSP90 activity, or with DMSO-vehicle. (E) Treatment of cells with 17-AAG at concentrations up to 2 μM has no effect on the steady-state levels of wild-type ChAT protein. Conversely, steady-state protein levels of P17A/P19A- (F) , V18M- (G) , and A513T-ChAT (H) are reduced following treatment of cells with 17-AAG compared to vehicle-control ( *** p ≤ 0.001; one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 4). (I) Proteasome inhibition by co-treatment with 5 μM MG132 for 18 h attenuates the effects of inhibition of HSP90 (1 μM 17-AAG, 24 h) on ChAT steady-state protein levels in ChAT-expressing SN56 cells. Control cells were treated with DMSO-vehicle. Proteasome inhibition was validated by immunoblotting for the accumulation of ubiquitinated cellular proteins ( n = 4). (J) Lysosomal inhibition by co-treatment with 50 μM chloroquine (CQ) for 18 h did not prevent the effects of 17-AAG treatment (1 μM, 24 h) on steady-state ChAT protein levels in SN56 cells. Lysosomal inhibition was validated by immunoblotting for the accumulation of the lysosome-associated protein LC3B-II ( n = 3).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Inhibition, Activity Assay, Mutagenesis, Western Blot, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSC/HSP70 and HSP90 activity enhances ChAT ubiquitination and reduces cellular ChAT enzymatic activity. (A) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for 24 h with 40 μM VER-155008 and for the final 6 h with 20 μM MG132 to inhibit degradation of ubiquitinated ChAT. Control cells were transfected with empty vector and/or treated with DMSO-vehicle. Inhibition of HSC/HSP70 by VER-155008 treatment enhanced ChAT ubiquitination, where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (B) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for either 8 or 24 h with 1 μM 17-AAG and for the final 6 h with 20 μM MG132. Inhibition of HSP90 by treatment with 1 μM 17-AAG for 8 h, but not for 24 h, enhanced ChAT ubiquitination where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (C) Cellular activity of wild-type ChAT is reduced following treatment of SN56 cells for 24 h with either 40 μM VER-155008 or 1 μM 17-AAG compared to vehicle-treated (DMSO) cells ( ** p ≤ 0.01). Treatment with VER-155008 trended toward a greater reduction in ChAT activity compared to 17-AAG ( p = 0.074; one-way ANOVA with Tukey's post-hoc test, mean ± SEM, n = 3).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

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

Journal: BMC Immunology

Article Title: Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

doi: 10.1186/1471-2172-6-24

Figure Lengend Snippet: Construction of soluble recombinant HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules . Structure and homogeneity of purified soluble HLA-DR*1101 recombinant molecules. (a) Schematic representation of HLA-DR*1101-Ig and HLA-DR*1101-Bir constructs. The extracellular region of HLA-DR*1101β is fused with the Basic Zipper (BZ) and His tag. The HLA-DRα chain is fused with the Acid Zipper (AZ) and the human (h)IgG1 constant region or the biotynilation site BirA, respectively. All constructs are cloned into the pMT/V5/His Drosophila expression vector, in frame with the Drosophila leader sequence Bip, under the control of a metallotioneine promoter, inducible by CuSO4 addition. The HLA-DR*1101-Ig molecule is secreted into the supernatant of Drosophila cells as dimer of HLA-DR*1101α/β heterodimers, because of a disulphide bond between two hIgG domains. (b) SDS-PAGE of HLA-DR*1101-Ig molecules secreted into the supernatant of S2 cells (sup) after CuSO4 induction, and after purification by protein A affinity chromatography (Prot A). Gels were run either in reducing (+βm) or non-reducing (-βm) conditions. Indicated are the bands corresponding to the migration of the HLA-DRα-hIg homodimers, HLA-DRα-hIg monomers and HLA-DR*1101β-His tag proteins. (c) Western blotting analysis of HLA-DR*1101-Ig molecules, separated on SDS-PAGE as shown in (b). The blotted filter was probed at the same time with the anti-His tag and anti-hIg probes, as indicated in figure. Empty circle and asterisk indicate the migration of the HLA-DRα-hIg and HLA-DR*1101β-His tag, respectively. (d) SDS-PAGE analysis of the HLA-DR*1101-Bir molecule contained into the supernatant (sup) of S2 cells after CuSO4 induction, and after purification by immunoaffinity chromatography with L243 mAb (L243).

Article Snippet: Protein production was monitored by dot blot analysis with either mouse anti-human HLA-DR α monoclonal antibody L243 (ATCC, Manassas, VA, USA) or rabbit anti-His tag polyclonal antibody His-probe (Santa Cruz, Santa Cruz, CA, USA), followed by an HRP-labelled goat anti mouse or anti rabbit antibody (Southern Biothecnology, Birmingham, AL, USA).

Techniques: Recombinant, Purification, Construct, Clone Assay, Expressing, Plasmid Preparation, Sequencing, Control, SDS Page, Affinity Chromatography, Migration, Western Blot, Chromatography

Journal: BMC Immunology

Article Title: Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

doi: 10.1186/1471-2172-6-24

Figure Lengend Snippet: Soluble recombinant HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules display different biochemical properties . Characterization of the native structure and stability of both soluble recombinant HLA-DR*1101 molecules. (a) Immunoprecipitation of HLA-DR*1101-Ig and HLA-DR*1101-Bir molecule with 30 μl of L243-sepharose beads from 1 ml of S2 cells culture supernatant after CuSO4 induction. The lanes contain the following material: 1. sup, 30 μl of culture supernatant before immunoprecipitation; 2. bound, the immunoprecipitated soluble recombinant HLA-DR*1101; and 3. not bound, 30 μl of culture supernatant after immunoprecipitation. Proteins were separated on SDS-page, transferred to filter and revealed with anti his-tag antibody. (b) Densitometric analysis of the protein bands displayed in panel (a), showing the elative efficiency of immunoprecipitation of the two soluble recombinant HLA-DR*1101 proteins with L243-Sepharose beads, as an indirect indicator of the percentage of correctly folded molecule. (c) Size exclusion chromatography of HLA-DR*1101-Ig molecules, after purification of ProtA affinity chromatography. The elution profile of the molecule from a Superdex 200 gel filtration column is shown. Inset shows the dot-blot analysis performed on the protein contained in the indicated elution peaks. Spotted proteins are probed with either anti-His tag antibody, to verify the presence of the HLA-DR*1101 molecule, or L243 mAb (Anti-HLA-DR) to verify the correct conformation of the molecule. (d) Elution profile from Superdex 200 gel filtration column and dot-blot analysis on eluted peaks of HLA-DR11-Bir molecules, performed as described in (c). (e) Calibration profile of the Superdex 200 gel filtration column.

Article Snippet: Protein production was monitored by dot blot analysis with either mouse anti-human HLA-DR α monoclonal antibody L243 (ATCC, Manassas, VA, USA) or rabbit anti-His tag polyclonal antibody His-probe (Santa Cruz, Santa Cruz, CA, USA), followed by an HRP-labelled goat anti mouse or anti rabbit antibody (Southern Biothecnology, Birmingham, AL, USA).

Techniques: Recombinant, Immunoprecipitation, SDS Page, Size-exclusion Chromatography, Purification, Affinity Chromatography, Filtration, Dot Blot

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of the N-terminal preproTRH(31–52) and C-terminal preproTRH(241–255) sequence deletion on precursor protein levels in AtT20 cells under steady state conditions. A, Western blot analysis of extracted peptides from AtT20 cells transfected with wild type (WT) proTRH, Δ31–52, or Δ241–255 mutants. After 48 h of transfection, levels of wild type (26 kDa) and mutated (∼23–24 kDa) prohormone levels were analyzed by densitometric analysis of the Western blot signals (n = 4, ±S.E.). Student's t test was done to compare each with wild type (*, p < 0.05). B, proTRH gene expression analysis. Transcript levels of wild type and Δ31–52 under steady state conditions were assayed by reverse transcription-PCR (n = 3; ± S.E.). Δ31–52 mRNA was transcript as much efficiently as than wild type. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, proTRH gene translation analysis. Protein translation for wild type and mutant Δ31–52 was performed using an in vitro cell free translation system (see “Experimental Procedures”). After protein immunoprecipitation,3H-labeled translation products were separated onto 12% SDS-PAGE polyacrylamide gels. The gels were sliced, and the cpm for each slice was counted. The peaks depicted in the graph represent the amount of prohormone (26 kDa) translated. Equal amount of protein was observed in both wild type and Δ31–52 (n = 4, ±S.E.). NC represents none DNA on the reaction.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Sequencing, Western Blot, Transfection, Gene Expression, Reverse Transcription, Mutagenesis, In Vitro, Immunoprecipitation, Labeling, SDS Page

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Analysis of Δ31–52 prohormone degradation by proteasome and lysosome systems. AtT20 cells transfected with wild type proTRH or Δ31–52 were treated with vehicle solution or proteasome inhibitor lactacystin (5 μm) for 16 h (A) or BFA (5 μg/ml) for 4 h (B). Equal amounts of protein from each sample were immunoblotted with anti-pCC10 antibody that recognizes the entire precursor protein (26 kDa for wild type (WT) and 23 kDa for Δ31–52 mutant) or anti-β-tubulin. NC represents AtT20 cells transfected with pEGFP-N1 plasmid as negative control. Protein levels were quantitated by AlphaEraseFC software. Graphics show the ratio between cells treated/untreated in wild type and mutant Δ31–52. Prohormone levels of Δ31–52 are recovered ∼2-fold by proteasomal inhibitor and BFA treatment (n = 4; ±S.E.). A representative blot from a single experiment is shown.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Negative Control, Software

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of specific mutations within the N-terminal proregion on protein abundance under steady state conditions in ATt20 and GH4C1. Wild type (WT) and PGL mutant DNAs were transfected into AtT20 (A) and GH4C1 (C) cells. NC indicates cells transfected with pEGFP-N1 plasmid. 48 h after transfection cells were lysed for immunoblotting with anti-pCC10. This antibody recognizes the precursor (∼26 kDa for ΔPGL, PGL/EKA, PGL/GGG, and PGL/AAA ∼23 kDa for Δ31–52), but it also recognizes N-terminal intermediate forms of the precursor (15 and 9.5 kDa). Precursor protein levels (26 kDa) are expressed as percentages of the levels in wild type cells. Values represent the mean ± S.E., n = 3. *, p < 0.05 relative to wild type by Student's t test. B, wild type, AVT, and R51G/R52G mutant DNAs were transfected into AtT20 cells, and the precursor protein levels were analyzed as described above (n = 3; ± S.E.). *, p < 0.05 relative to wild type by Student's t test. NC, negative control.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Quantitative Proteomics, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Negative Control

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Analysis of proTRH-derived peptides secretion in wild type and N-terminal mutants. N-terminal (pEH24) (A), C-terminal (5.4 kDa) (B), and TRH (C) proTRH derived peptides from 1-h basal release (BR), 1-h stimulated release (SR) with 1 mm BaCl2, and cell content (CC) were measured by radioimmunoassay in AtT20 cell expressing wild type (WT) and mutants Δ31–52, PGL/EKA, ΔPGL, PGL/GGG, PGL/AAA, R51G/R52G, and AVT/GGG. Peptides levels in each fraction are expressed as percentage of the levels in wild type cells (100%) (n = 3; ± S.E.). *, p < 0.05 relative to wild type by Student's t test. See Table 1 for a description of mutants.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Derivative Assay, RIA Assay, Expressing

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of proteasome inhibition in wild type (WT), ΔPGL, and PGL/GGG on precursor protein levels. AtT20 cells expressing ΔPGL (A) or PGL/GGG (B) mutants and AtT20 cells expressing wild type preproTRH were exposed to vehicle solution (–) or 5 μm lactacystin (+) for 16 h. NC represents AtT20 cells transfected with pEGFP-N1 plasmid. Cells extracts were prepared, and equal amounts of protein (30 μg) were analyzed by Western blotting with anti pCC10. Protein recovered is expressed as the ratio lactacystin+/lactacystin–. Inhibition of the proteasome induced 2-fold recovered ΔPGL precursor compared with wild type (A) but was 1.3-fold lower for PGL/GGG precursor (n = 4, ±S.E.). NC, negative control.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot, Negative Control

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of ER transport with Brefeldin A in wild type, ΔPGL, and PGL/GGG on precursor protein levels. AtT20 cells expressing ΔPGL (A) or PGL/GGG (B) mutants and AtT20 cells expressing wild type (WT) preproTRH were exposed to vehicle solution (–) or 5 μg/ml BFA (+) for 4 h. Cells extracts were prepared, and equal amounts of protein (30 μg) were analyzed by Western blotting with anti pCC10. Protein recovered was expressed as the ratio BFA+/BFA–. For 4 h of incubation, BFA proportionally induces an equal accumulation in wild type and both ΔPGL and PGL/GGG precursors (n = 4, ±S.E.).

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Expressing, Western Blot, Incubation

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Determination of wild type proTRH, ΔPGL, and PGL/GGG precursor half-lives by pulse-chase experiments. 48 h after AtT20 cells transfection with wild type (WT), ΔPGL, and PGL/GGG DNA constructs, protein precursors were radiolabeled for 2 h with [3H]leucine, and protein levels (cpm) were followed at 0-, 45-, and 90-min chase times. Graphics show the evolution of proTRH precursor (26 kDa) through 90 min. The results are the means ± S.E. (n = 3).

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Pulse Chase, Transfection, Construct