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: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: International journal of oncology

Article Title: Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

doi: 10.3892/ijo.2014.2397

Figure Lengend Snippet: Figure 3. Reduced production of VEGF and transcription of both VEGF-A and VEGF-B by C. militaris extract. (A) Dot blot-human angiogenesis array of MeWo culture medium after C. militaris extract or cordycepin treatment. Culture media of MeWo cells incubated with control, 5% C. militaris extract, 20 µM cordycepin, or 100 µM cordycepin for 48 h were used to detect the production of various angiogenesis-related proteins. All treatment groups showed lower production of angiogenin, TIMP-1, PDGF-AA, and VEGF compared to the control group. Production of TIMP-1 and VEGF was especially reduced in the 5% C. militaris treatment group. (B and C) qPCR of VEGF-A and VEGF-B. Transcription levels of VEGF-A and VEGF-B were quantified from MeWo cells after incubation with either 5% C. militaris extract (B) or 20 µM cordycepin (C) for 48 h. Transcription levels of VEGF-A and VEGF-B were significantly decreased after treatment with 5% C. militaris extract, while only VEGF-A was decreased after treatment with 20 µM cordycepin. *P<0.05 significantly different from the control group. ns, not significant.

Article Snippet: Screening for angiogenesis-related factors produced by MeWo cells was carried out by using the Human angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA).

Techniques: Dot Blot, Incubation, Control

Journal: International journal of oncology

Article Title: Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

doi: 10.3892/ijo.2014.2397

Figure Lengend Snippet: Figure 6. Treatment with C. militaris extract reduced angiogenesis. (A) Decrease of FITC-lectin intensity after treatment with 5% C. militaris extract. Using the Cultrex Directed In Vivo Angiogenesis Assay (DIVAA) (Trevigen, Gaithersburg, MD, USA), angioreactors containing a mixture of BME and DDW or BME and 5% C. militaris extract were implanted subcutaneously into the flanks of mice for 16 days. After extraction, FITC-lectin intensity was measured using excitation 485 nm, emission 510 nm. (B) Chorioallantoic membrane (CAM) assay showed that neovascularization was inhibited by C. militaris extract. Eight days post-fertilized chicken egg shells were cut to place silicon rings on the CAM. The inner spaces of the silicon rings were filled with DDW or 50 µl of C. militaris extract. Egg shells were covered using transparent tape, and eggs were incubated for 48 h. Results were observed by decrease or increase of vascular branch formation from main capillary vessels.

Article Snippet: Screening for angiogenesis-related factors produced by MeWo cells was carried out by using the Human angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vivo, Angiogenesis Assay, Extraction, Membrane, Chick Chorioallantoic Membrane Assay, Incubation

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein binds to and enters the cell through STEAP2 (A) Comparison of the cell-binding capacity of SARS-CoV-2 wild type (WT) N protein and Omicron N protein expressed in either E. coli or mammalian cells. 1 × 10 5 A549 cells were used to mixed with 1 μg WT N or Omicron N proteins. One hour after protein addition, allophycocyanin (APC) conjugated anti-His antibody was used to detect the cell binding capacity of WT N protein or Omicron N protein. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). (B) Antibody blocking assay. Aliquots of 10 μg of SARS-CoV-2 N protein were pre-mixed with 0, 1, 3, 10, 30, and 100 μg of normal mouse IgG or anti-N monoclonal antibody (NP-mAb-40) and incubated at 4°C overnight. The antibody/N protein complex was used for the A549 cell surface binding assay. The blocking capacity of anti-N antibody was normalized to N protein only control. (C) Membrane fractions of A549 and HPAEpiC cells were extracted and incubated with N protein conjugated beads for 3 h binding at 4°C, and pull-downed for LC-MS-MS analysis (upper panels). A549 and HPAEpiC cells were suspended and treated with N protein for 1 h on ice. After incubation, cells were crosslinked with 3 mM DTSSP for 1.5 h. Then, cells were lysed in RIPA lysis buffer, and N protein complex in the lysate was immunoprecipitated for LC-MS-MS analysis (lower panels). Y axis denotes −logP values while the X axis shows log2 fold change values. Orange dots highlight the statistically significant proteins, with p value < 0.05 (-Log p > 1.3) and fold change>2, and the enriched plasma membrane protein was labeled on the plot. Identified proteins were further sorted by HuMemProtDB. (D) To knock-down (KD) STEAP2 expression, HPAEpiC cells were infected with lentivirus carrying STEAP2 shRNA followed by puromycin selection for 14 days. The STEAP2 mRNA expression levels were assessed by qRT-PCR, and the relative KD efficiency of shSTEAP2 was compared to shLacZ control (left-hand side panel). N protein binding capabilities to HPAEpiC STEAP2 KD cells and shLacZ control KD cells were assessed by flow cytometry analysis, and data were shown as mean fluorescence intensity (MFI). (right-hand side panel). (E) Western blot analysis of STEAP2 in wild type (WT) and knock-out (KO) A549 cells were shown. N protein binding to A549 STEAP2 KO cells was assessed by flow cytometry analysis and shown as mean fluorescence intensity (MFI). Ccr (crotonyl-CoAcarboxylase/reductase, a bacterial protein) binding was used as a control. (F) SARS-CoV-2 N protein enters alveolar cells. HPAEpiC cells were treated with 10 μg SARS-CoV-2 N protein overnight and then stained with anti-N antibody. The localization of N protein (Red) was checked by fluorescence microscope and cell morphology was observed by dimensional interference contrast (DIC). Nuclei of cells were stained by DAPI (blue). (G) N protein entering cells by endocytosis and N protein co-localization with STEAP2. HPAEpiC alveolar cells were seeded on 8 well slides. Cells were pretreated with endocytosis inhibitors HCQ, or Dynasore. Then the cells were treated with N protein overnight. After treatment, the cells were stained by specific antibodies to detected N protein (red), endosome marker (EEA1) (green), and STEAP2 (yellow). Cells were observed under fluorescent microscopy (Invitrogen tech.). Scale bar: 50 μm. All data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; t test. See also Figure S5 .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Comparison, Binding Assay, Flow Cytometry, Fluorescence, Antibody Blocking Assay, Incubation, Blocking Assay, Control, Membrane, Liquid Chromatography with Mass Spectroscopy, Lysis, Immunoprecipitation, Clinical Proteomics, Labeling, Knockdown, Expressing, Infection, shRNA, Selection, Quantitative RT-PCR, Protein Binding, Western Blot, Knock-Out, Staining, Microscopy, Marker

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein delivers nucleic acids into cells (A) N protein-RNA complex binding to the cell surface. Aliquots of 10 μg SARS-CoV-2 N protein were incubated with 1 μg of indicated RNAs for 1 h at 4°C, and added to A549 or HPAEpiC cultures. SARS-CoV-2 N protein only without RNA was used as a control. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; t test. (B) The observation of N protein-RNA enters into cells. HPAEpiC were seeded onto 8-well glass slides (40,000 cells/well). SARS-CoV-2 N protein 10 μg and 40 μg RNA-FAM (green) were mixed for 1 h at 4°C. cells were treated with SARS-CoV-2 N-RNA-FAM mixture for 1 h. The groups of non-treated cells and RNA-FAM only were as controls. After treatment, N protein was detected by anti-N antibody (Red). The localization of RNA-FAM was green. DAPI (blue) indicates cell nuclei. Scale bar: 15 μm. (C) Lattice light sheet microscopy time lapse imaging of N protein-RNA complex entering into HPAEpiC cells. SARS-CoV-2 N protein 10 μg was mixed with 40 μg RNA-FAM (fluorescein) for 1 h at 4°C and then treated with ice-cooled alveolar cells. The signals of RNA-FAM and Hochest 33,342 were monitored by lattice light sheet microscopy at different time points.

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Binding Assay, Incubation, Control, Flow Cytometry, Fluorescence, Microscopy, Imaging

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein-assisted nucleic acid dispersion and expression in the co-culture environment (A)The co-culture system consisted of A549 as recipient cells, and 293T pre-transfected with two plasmids, one expressing GFP and the other expressing SARS-CoV-2 N protein or the pcDNA3.1 empty vector. (B–E) After 24 h co-culture of the donor cells and recipient cells, cell pool was stained with cytokeratin 18 (an A549 marker) and SV40 large T antigen (a 293T marker). A549 cells in the cell pool were gated from cytokeratin 18 positive and large T antigen negative. A549 GFP positive percentage was further assessed by flow cytometry analysis. Effects of SARS-CoV-2 N variants (B), treatment with RANTES (C), the p38 inhibitor SB203580 (D), or anti-N neutralizing antibody (E) were accessed by adding these effectors to the medium. Experiments are performed in three to five biological replicates. ∗, p value <0.05 (paired two-tailed student’s t -test). (F) SARS-CoV-2 N protein promotes gene delivery by cell-free diffusion to neighboring cells. A549 cells were plated in the lower chamber, while 293T donor cells co-transfected with plasmids expressing EGFP and indicated N proteins in the upper chamber. After 3 days of co-culture, GFP positive A549 cells were observed and counted. See also Figures S10 and .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Dispersion, Expressing, Co-Culture Assay, Transfection, Plasmid Preparation, Staining, Marker, Flow Cytometry, Two Tailed Test, Diffusion-based Assay

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Bioprocessing, Recombinant, Magnetic Beads, Protease Inhibitor, Sequencing, Modification, SYBR Green Assay, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: N-glycosylation acts as a switch for FGFR1 trafficking between the plasma membrane and nuclear envelope

doi: 10.1186/s12964-023-01203-3

Figure Lengend Snippet: Hypothetical model of the N-glycosylation-dependent cellular trafficking of FGFR1. A . After co-translational synthesis in the ER, the wild type FGFR1 is N-glycosylated at several positions. The N227 site precludes FGFR1 transport to the nuclear envelope, while N-glycosylation sites in the D2 and D3 domain promote FGFR1 transport via the ER/Golgi/secretory vesicles to the plasma membrane, where the receptor becomes available for FGFs’ stimulation. During the transport to the cell surface, N-glycosylation of FGFR1 ensures low level of FGFR1 autoactivation in the intracellular compartments in the absence of FGFs. B . Signal peptide (SP)-driven co-translational ER targeting of the N-glycosylation-deficient FGFR1 (FGFR1.GF) results in the initial accumulation of FGFR1.GF in the ER, where it binds several protein folding and quality control factors, such as BiP or protein disulfide isomerase A4. In the absence of N-glycans, the extracellular region of FGFR1.GF undergoes unfolding and aggregation, initiating ligand-independent FGFR1.GF autoactivation. Alternatively, the absence of N-glycans in the properly folded extracellular region of FGFR1.GF facilitates FGFR1.GF dimerization and activation in the absence of FGFs. In both scenarios, intracellular FGFR1 displays a high degree of autoactivation. Lateral diffusion of the ER-trapped FGFR1.GF within the ER-membrane, which is continuous with the nuclear envelope, results in the transport of FGFR1.GF to the nuclear envelope. Importins and NPC are likely involved in this step. FGFR1.GF is retained in the nuclear envelope presumably by participating in complexes with a precise set of nuclear proteins. Importantly, FGFR1.GF localized to the nuclear envelope is highly kinase active, indicating the presence of a novel nuclear FGFR1 signaling cascade

Article Snippet: The antibodies directed against COPB (#sc-393615), FGF-2 (#sc-74412), heterogenous nuclear ribonucleoproteins C1/C2 (#sc-32308), HSP90 (#sc-13119), U4/U6.U5 tri-snRNP-associated protein 1 (#sc-376460), protein disulfide isomerase A4 (#sc-390530), nucleolin (#sc-8031), SBP-tag (#sc-101595) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Control, Activation Assay, Diffusion-based Assay

Journal: Cell reports

Article Title: Integrated genomic and proteomic analyses identify stimulus-dependent molecular changes associated with distinct modes of skeletal muscle atrophy

doi: 10.1016/j.celrep.2021.109971

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-alpha- tubulin (11H10) , Cell Signaling Technologies , 2125S; RRID:AB_2619646.

Techniques: Recombinant, SYBR Green Assay, Membrane, Electron Microscopy, Bicinchoninic Acid Protein Assay, Cell Based Assay, Reverse Transcription, Muscles, Gene Expression, Control, Mass Spectrometry, Software

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: HT-29 cells and HCPECs were stimulated with LPS (200 ng/ml) at various time points as indicated. ( A ) Representative western blot analysis of HT-29 cells. Phosphorylation and/or total protein levels of ERK3, ERK4, MK5 and JNK were monitored. Tubulin immunoblots and Ponceau S staining were employed to monitor equal loading. ( B ) Changes in the expression and phosphorylation of ERK3 protein are shown as a fold change after normalization with internal loading control. Each time point was normalized to unstimulated cells (0). Fold change values from three independent experiments (n = 3) are represented as mean ± SEM. ( C ) Activation kinetics of MK5 in HT-29 cells stimulated with LPS. Fold change in MK5 phosphorylation levels upon LPS stimulation normalized to total protein levels and expression levels of MK5 normalized to internal loading control are shown. Fold change values from three independent experiments (n = 3) are represented as mean ± SEM. ( D ) Quantitative RT-PCR analysis of ERK3 expression. Each biological replicate was measured in triplicates. Log2 fold change in gene expression is presented as mean ± SEM of three independent experiments (n = 3); *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Turkey’s post-test. ( E ) ERK3 protein stability was assessed by CHX chase at 0 hr, 0.5 hr, 1 hr, 2 hr, 3/4 hr and 6 hr in the presence and absence of LPS (30 min pre-treatment). Western blot analyses were performed and representative results are presented. ERK3 protein levels in respect to the untreated cells (-LPS, 0 hr) were calculated using ImageJ and data are presented as mean fold change ± SEM from three independent experiments (n = 3). ( F ) Graph presents ERK3 protein levels quantified in respect to the untreated cells (0) of unstimulated (-LPS) and LPS stimulated (+LPS) cells, respectively and data are presented as mean fold change ± SEM from three independent experiments (n = 3). ( G ) HCPECs were stimulated with LPS and immunoblot analyses of the phosphorylation and/or total protein levels of ERK3, MK5 and p38 were performed. Actin and Ponceau S staining were used as loading controls. ( H ) Plotted here are fold changes in expression of ERK3 protein. Results are shown as mean ± SEM fold change after normalization with the levels of internal loading control. Each time point was normalized in respect to unstimulated HCPECs (0). Data are a representative of three independent experiments (n = 3); *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Turkey’s post-test. ( I-K ) Plotted here are fold changes in the phosphorylation of ( I ) MK5 at T182, ( J ) ERK1/2 and ( K ) p38 in response to LPS stimulation normalized to the respective total protein levels as well as the expression levels of total proteins normalized in respect to the internal loading control. Each time point was normalized in respect to the unstimulated cells (0). Fold change values are presented as mean ± SEM from three independent experiments (n = 3). ( L ) Quantitative RT-PCR analysis of ERK3 mRNA expression levels. Log2 fold change in gene expression is presented as mean ± SEM of three independent experiments (n = 3); *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Turkey’s post-test. ( M-N ) LPS-mediated ubiquitination of endogenous ERK3 in ( M ) HT-29 cells and ( N ) HCPECs. HT-29 cells and HCPECs were seeded and treated as mentioned in the Materials and methods. Total cell lysates (TCL) and endogenous ERK3 immunoprecipitates (IP) were analyzed by immunoblotting. Levels of ERK3 and polyubiqutination were monitored. Actin and Ponceau S staining were used as loading controls for TCL western blot analysis. Results are representatives of at least two experiments showing the same tendency. ERK3 kinetics in response to other immune stimuli are presented in ). Figure 2—source data 1. Full membrane scans for western blot images for .

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Phospho-proteomics, Staining, Expressing, Control, Activation Assay, Quantitative RT-PCR, Gene Expression, Ubiquitin Proteomics, Membrane

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: ( A–B ) Western blot analysis of CaCo2 cells stimulated with LPS. CaCo2 cells were stimulated at indicated time points with LPS and activation status of ERK3 as well as the phosphorylation of p38 were analyzed by immunoblotting. Actin and Ponceau S staining were used as loading controls. ( A ) Representative western blot. ( B ) Changes in the expression and phosphorylation of ERK3 protein are shown as a fold change after normalization with internal loading control. Each time point was normalized to unstimulated cells. Fold change values from two (n = 2) independent experiments are represented and means were connected for better visualization. Figure 2—figure supplement 1—source data 1. Full membrane scans for western blot images for .

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Staining, Expressing, Control, Membrane

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: ( A-F ) HCPECs and HT-29 cells were stimulated for 0, 0.5, 1 and 2 hr with IL-1β, Pam3CSK4 and R848, cells were lysed for western blot analysis. Representative immunoblots depicting phosphorylation and total protein levels of ERK3 (S189)/ERK3 as well as the phosphorylation of p38 MAPK. Ponceau S staining and actin were used as loading controls.

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Phospho-proteomics, Staining

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: ( A–D ) HCPECs were transfected with siRNA targeting ERK3 (siERK3) or control (siCo), 24 hr post-transfection, medium was exchanged for MEM without supplements and cells were stimulated with IL-1β or Pam3CSK4 for 24 hr. Supernatants were collected for IL-8 ELISA analysis and cells were subjected to immunoblotting. Levels of IL-8 are presented as mean concentration in pg/ml ± SEM from two biological replicates (n = 2). Knockdown efficiency was determined using ERK3 antibody and phosphorylation status of p38 and ERK1/2 was monitored. Actin and Ponceau S staining were used as loading controls. ( E-H ) Control (shCo) and ERK3-depleted (shERK3) HT-29 cells were seeded in 12-well plate. After cells reached 70–80% confluency, medium was exchanged for McCoy’s without serum and cells were stimulated with IL-1β or Pam3CSK4 for 24 hr. Supernatants were collected for IL-8 ELISA analysis and cells were subjected to western blot analysis. IL-8 concentration in pg/ml is presented as mean ± SEM of two biological replicates (n = 2). Expression levels of ERK3 were verified to determine knockdown efficiency and phosphorylation of p38 and ERK1/2 was monitored. Actin and Ponceau S staining are provided as loading controls. Figure 4—figure supplement 3—source data 1. Full membrane scans for western blot images for .

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Concentration Assay, Knockdown, Phospho-proteomics, Staining, Expressing, Membrane

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet:

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Concentration Assay, Recombinant, Sequencing, shRNA, CRISPR, Mutagenesis, Enzyme-linked Immunosorbent Assay, Cell Fractionation, Ab Array, Extraction, Activation Assay, Luciferase, Chemotaxis Assay, Neutralization, Control, Software, Staining, Fluorescence

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Definition of the earliest PanINs and their physical location in the pancreas of KC mice. A and F) The representative 3D imaging projection of the whole pancreas of 2 (A)‐ and 4 (F)‐week‐old Pdx1 ‐Cre; LSL‐ Kras G12D/+ (KC) transgenic mouse using 3D histological analysis. Red arrow: the early PanIN. Blue signal: the nucleus staining; White signal: CK19‐staining; Red signal: the blood vessel staining. Scale bar: 1 mm. B and G) Quantification of PanIN number in 2 (B)‐ and 4 (G)‐week‐old transgenic mice. Each dot represents the datum of one mouse. Values were presented as mean ± SD. C and H) The distribution of the earliest PanIN lesions from 45 and 24 of 2 (C)‐ and 4 (H) ‐week‐old KC mice, respectively. One yellow dot indicates one lesion. n = 109 (C); n = 211 (H). D and I) Stacked bar plot showing the percentage of PanIN in the pancreas's head, body, and tail of 2 (D)‐ and 4 (I)‐week‐old KC mice. E and J) Quantification of PanIN and islet association in the pancreas of 2 (E)‐ and 4 (J) ‐week‐old KC mice. An association is defined by the distance between lesion and islet within 300 µm.

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Imaging, Transgenic Assay, Staining

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Genetic alterations in the earliest PanIN and Muc4 up‐regulation cooperate with oncogenic Kras G12D for PanIN initiation. A) The most common genetic alterations in the earliest PanINs from the pancreas of 2‐week‐old KC mice. The data analyzed in this study were obtained from 21 lesions in 13 mice, which were compared to the normal pancreatic region within the same KC mouse. In addition, two control mice were included for comparison. To ensure accuracy, all mouse‐specific single nucleotide polymorphisms (SNPs) were excluded by referencing the UCSC Genome Browser website. B) The RNA‐seq data of the Muc4 gene and Sirpb1a , comparing the PanIN sample with the control sample from a 2‐week‐old KC mouse (M1376). C) The IHC staining of Ki67 proliferation marker. Representative images (left panel) and quantification (right panel) of Ki67 staining in 4‐week‐old control and KC mice. Bar, 50 µm. Each dot represents the datum of one mouse. Values were presented as mean ± SD, n = 11 mice. *, P <0.05; **, P <0.01; ***, P < 0.001 (two‐tailed Student's t‐test). D) Plots of the survival probability of pancreatic cancer patients with MUC4 (left panel) and SIRPB1 (right panel) mRNA expression levels from the Human Protein Atlas as shown in methods. P values are calculated by log‐rank test. E–H) The Alcian Blue staining and IHC analysis with anti‐Muc4 antibody in 4‐week‐old control and KC mice. Representative images of IHC staining with Alcain Blue and Muc4 E), quantification of Alcain Blue F) and Muc4 G) in the early PanIN cells. Bar, 50 µm. The H score of Muc4 expression in 4‐week‐old control and KC mice H). Each dot represents the datum of one mouse. Values were presented as mean ± SD, n = 11 mice. ****, P < 0.0001 (two‐tailed Student's t‐test). I and J) EpCAM + /Muc4 − ‐ or double EpCAM/Muc4‐positive pancreatic cells isolated by FACS from 4‐week‐old KC mice for spheroid formation analysis I) and soft agar colony formation analysis J). N indicates independent experiments, and n indicates total measurements in all experiments. Each dot represents the datum from one measurement. Values show mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). K and L) Primary acinar cells were isolated from 4‐week‐old control or KC mice for spheroid formation analysis K) and soft agar colony formation analysis L). N indicates independent experiments, and n indicates total measurements. Each dot represents the datum from one measurement. Values show mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Control, Comparison, RNA Sequencing, Immunohistochemistry, Marker, Staining, Two Tailed Test, Expressing, Isolation

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: αSMA + fibroblasts associate with the earliest PanIN cells to promote Kras G12D/+ pancreatic cell transformation and stemness properties. A) Representative image of 3D histology‐detected the earliest PanINs in the whole pancreas of 2‐week‐old KC mice. Blue signal: the nucleus staining; White signal: CK19‐staining (PanIN cells); Green signal: αSMA staining (activated fibroblasts). Bar, 100 µm. B) Quantification of the percentage of the earliest PanINs associated with αSMA + fibroblasts. C) Representative images of the IHC staining of fibroblasts with anti‐αSMA antibody in 4‐week‐old KC mice (left panel) and quantification of the percentage of early PanINs associated with αSMA + fibroblasts (right panel). Bar, 50 µm. D) Close contact with fibroblasts promotes acinar‐to‐ductal metaplasia of Kras G12D/+ pancreatic acinar cells in 3D Matrigel coculture systems. X: Kras G12D/+ pancreatic acinar cells only (1000 cells). F: coculture with Kras +/+ fibroblasts (2000 cells). I: coculture with Kras +/+ islet cells (2000 cells). Upper panel: Representative images in coculture experiment in Kras G12D/+ pancreatic cells. Bar, 50 µm. Bottom panel: Quantification of cyst number. E and F) Close contact of Kras G12D/+ pancreatic acinar cells with fibroblasts promotes sphere formation. E) 1000 Kras G12D/+ pancreatic acinar cells labeled with CellTracker Green CMFDA were cocultured with 2000 Kras +/+ fibroblasts or 2000 Kras +/+ isle cells for 14 days in a 96‐well low attached plate. (Left panel) Representative images of the cocultured Kras G12D/+ pancreatic cells. (Right panel) Quantification of sphere number. Spheres with a diameter ≥ 100 µm were counted. F) 1000 Kras G12D/+ pancreatic acinar cells labeled with CellTracker Green CMFDA were cocultured with different ratios of Kras +/+ fibroblasts for 14 days in a 96‐well low attached plate. Spheres with a diameter ≥ 100 µm were counted. In the above experiments, N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). G) EpCAM + /PDGFRα + cell clusters derived from the pancreas of 4‐week‐old control or KC mice by MACS dissociation and FACS with EpCAM/PDGFRα antibodies for cyst formation analysis. Representative images of cysts without and with lumen were shown in the left panel. CK19 staining indicates pancreatic ductal cells, and αSMA staining indicates activated fibroblasts. Bar, 100 µm. The number of cysts was counted and compared in the right panel. N indicates independent experiment and n indicates cyst number. H) The same cell clusters as above, in addition two more controls including EpCAM + and non‐ EpCAM + /PDGFRα + clusters from KC mice, were used for sphere formation analysis. Representative images of spheres were shown in the upper panel and the number of spheres were counted (lower panel). Spheres with a diameter ≥ 100 µm were counted. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ***, P < 0.001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Transformation Assay, Staining, Immunohistochemistry, Labeling, Two Tailed Test, Derivative Assay, Control

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Muc4 overexpression in Kras G12D/+ pancreatic cells promotes fibroblast activation and recruitment in the earliest PanIN. A) The mRNA levels of Muc4, Muc1, and Muc5a in the EpCAM + /PDGFRα + clusters derived from the pancreas of 4‐week‐old KC mice were measured and compared with that of the control mice after normalized with the control gene, GAPDH . Primary mPSCs in all experiments were isolated from the pancreas of 4‐week‐old control mice. For chemotaxis analysis, mPSCs were labeled with CellTracker Green CMFDA for 10 mins before the experiment. B) Expression of ACTA2 gene in mPSC treated with conditional media derived from EpCAM + /Muc4 − or EpCAM + /Muc4 + pancreatic cells isolated by FACS from 4‐week‐old control or KC mice by qPCR. C) Left panel: Representative images of µslide chemotaxis analysis of mPSC after treated with conditional media as B). Right panel: Quantitation of the percentage of chemotaxis. D–F) EpCAM + /Muc4 − ‐ or EpCAM + /Muc4 + ‐pancreatic cells were cocultured with mPSC cells for cyst formation analysis D), sphere formation analysis E), and soft colony formation analysis F). For sphere formation analysis, sphere with a diameter ≥ 100 µm were counted. G) Expression of ACTA2 gene in mPSC treated with conditional media derived from pancreatic acinar cells isolated from 4‐week‐old control or KC mice by qPCR. These primary pancreatic acinar cells were infected with lentiviral GFP or lentiviral GFP‐MUC4 (MUC4/X). H) Left panel: Representative images of µslide chemotaxis analysis of mPSC after treated with conditional media as G). Right panel: Quantitation of the percentage of chemotaxis. I and J) Pancreatic acinar cells from KC or control mice were ectopically expressed GFP‐MUC4/X or GFP only and cocultured with mPSC cells for sphere formation analysis I) and soft colony formation analysis J). For soft agar colony formation analysis, the colony with a diameter ≥ 50 µm was counted. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. **, P < 0.01; ****, P < 0.0001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Over Expression, Activation Assay, Derivative Assay, Control, Isolation, Chemotaxis Assay, Labeling, Expressing, Quantitation Assay, Infection, Two Tailed Test

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Activin A from Muc4 overexpressed and Kras G12D/+ pancreatic cells facilitate fibroblast recruitment for PanIN formation. A) Cytokines analysis of conditional media from the cell clusters isolated from 4‐week‐old control or KC mice using RayBio® Mouse Biotin‐Label Based Antibody Array (Mouse L‐308 Array, Glass Slide). B) The effect of Activin A on mPSC chemotaxis was analyzed using μ‐Slide chemotaxis analysis. Representative images (left panel) and quantification (right panel) of the chemotaxis effect. C) Quantification of Activin A in the conditional media of EpCAM + /Muc4 − ‐ or EpCAM + /Muc4 + ‐pancreatic cells using ELISA analysis. D) Anti‐Activin A antibody abolishes mPSC chemotaxis. Conditional media from EpCAM + /Muc4 + ‐pancreatic cells were pre‐treated with or without 4 µg mL −1 of anti‐Activin A antibody for 30 mins and were subjected to µslide chemotaxis analysis. E) Quantification of Activin A in the conditional media of pancreatic acinar cells infected with lentiviral GFP or lentiviral GFP‐MUC4 (MUC4/X) using ELISA. F) Anti‐Activin A antibody abolishes mPSC chemotaxis. Conditional media from lentiviral GFP‐MUC4/X‐infected pancreatic acinar cells were pre‐treated with or without 4 µg mL −1 of anti‐Activin A antibody for 30 min and were subjected to μ‐Slide chemotaxis analysis. G) Increases of Activin A secretion of media from FACS‐isolated pancreatic cells cocultured with or without fibroblasts by ELISA analysis. H) Activin A secretion from Kras G12D/+ pancreatic acinar cells infected with the indicated lentivirus after cocultured with or without fibroblasts by ELISA analysis. In the above experiments, N indicates independent experiments, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two‐tailed Student's t‐test). I) Activin A mRNA expression in PanIN and PanIN‐associated fibroblasts in 4‐week‐old KC mice was detected by an Opal Multiplex IHC Assay. Representative IHC images (left panel) and quantification of Activin A expressed in early PanIN cells (right panel). Bar, 20 µm. White signal: DAPI; Cyan signal: Normal duct cells and PanIN cells stained with anti‐CK19 antibody; Red signal: αSMA + fibroblasts stained with anti‐αSMA antibody; Green dot signal: Activin A mRNA with dig‐labeled antisense mRNA probes. Values were presented as mean ± SD, PanIN lesion n = 29 from 12 KC mice. J) Sera from 4‐week‐old control or KC mice were used to perform Activin A ELISA analysis. N = 7 mice; two duplicate experiments for each mouse, n = total 14 repeated measurements. Each dot represents the datum from one measurement. Values were presented as mean ± SD. *, P < 0.05 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Isolation, Control, Ab Array, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Expressing, Multiplex Assay, Staining, Labeling

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: The proposed model for the cooperation of oncogenic Kras G12D/+ , Muc4 overexpression, and fibroblast activation for PanIN initiation. A–E) 3‐week‐old KC mice were administered either Saline solution or a dose of 1 µg k −1 g follistatin (FST) for a duration of two weeks. After the two‐week treatment period, we performed integrated 3D/2D pancreas histology to assess fibroblast recruitment and PanIN formation. A) Representative images depicting 3D and 2D histology of the pancreas. In the 3D staining images, green color represents CK19 staining, indicating PanIN or ductal cells; red color represents αSMA staining, indicating activated fibroblasts; and white color represents DAPI staining, indicating nuclei. In the 2D IHC images, brown color represents αSMA staining, indicating activated fibroblasts. Scale bar: 100 µm. B) The percentage of lesions associated with fibroblast. Each dot represents the datum of one mouse. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. ***, P < 0.001 (two‐tailed Student's t‐test). C) The H score of αSMA + fibroblasts associated with lesion of specified mice. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. **, P < 0.01 (two‐tailed Student's t‐test). D) Representative lesion images (upper panel) and atrophy lesion quantification (lower panel) at KC mice treated with Saline solution or FST. Green color represents CK19 staining, indicating PanIN. Each dot represents the datum of one mouse. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. **, P < 0.01 (two‐tailed Student's t‐test). E) The percentage of lobe and lesions volume in KC mice treated with Saline solution or FST. Mouse number and lesion number are denoted by N and n, respectively. The total tissue volume calculated in the Saline solution group is 279.4 mm 3 , while in the FST group, it is 252.8 mm 3 . F) mPSCs were obtained from 4‐week‐old control mice and subjected to overnight infection with 10 MOI of the specified lentiviral shRNA. After one day of recovery from the virus infection, we selectively enriched lentiviral shRNA‐positive cells by applying puromycin selection at a concentration of 1 µg mL −1 for three days. Following a two‐day recovery period, the cells were subjected to Western blot analysis (upper panel) and cocultured with double EpCAM and Muc4‐positive pancreatic cells isolated from 4‐week‐old KC mice for the assessment of soft agar colony formation (lower panel). The number of colonies larger than 50 µm was quantified after a 14‐day coculture period. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). G) Proposed model of oncogenic Kras G12D ‐mediated PanIN initiation. The overexpression of Muc4 (Muc4/X) in Kras G12D/+ pancreatic cells enhances cell transformation and stimulates Activin A secretion, which in turn activates and recruits' fibroblasts. The activated fibroblasts further promotes Activin A secretion, elevates pancreatic cell transforming status, and enhances cancer stemness properties, thereby contributing to PanIN initiation. The inhibition of Activin A signaling using Follistatin (FST), an Activin A antagonist, effectively suppresses fibroblast recruitment and inhibits PanIN formation. Additionally, reducing Activin A expression in mPSCs through the use of lentiviral Inhba shRNA also impedes PanIN formation.

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Over Expression, Activation Assay, Saline, Staining, Two Tailed Test, Control, Infection, shRNA, Virus, Selection, Concentration Assay, Western Blot, Isolation, Transformation Assay, Inhibition, Expressing

Journal: Developmental cell

Article Title: SUPPRESSING MITOCHONDRIAL RESPIRATION IS CRITICAL FOR HYPOXIA TOLERANCE IN THE FETAL GROWTH PLATE

doi: 10.1016/j.devcel.2019.04.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti LDHA , Cell Signaling Technology , Cat# 2012, RRID:AB_2137173.

Techniques: Virus, Luciferase, Plasmid Preparation, Recombinant, Saline, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Marker, Western Blot, Stripping, In Situ, Imaging, Live Cell Imaging, Membrane, Cell Based Assay, Software