Journal: Cell

Article Title: Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells

doi: 10.1016/j.cell.2022.04.039

Figure Lengend Snippet:

Article Snippet: Anti-human CD184 , Miltenyi Biotec , Cat# 130-117-519; RRID:AB_2734059.

Techniques: Blocking Assay, Plasmid Preparation, Recombinant, Electroporation, CRISPR, Adjuvant, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Cell Culture, Gene Expression

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression attenuates the effects of TGFβ1 on normal and TAO OFs. ( A – G ) Healthy or TAO OFs were transfected with Epac1 -overexpressing plasmid ( Epac1 ), treated with TGFβ1 (10 ng/mL), and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); relative α-SMA and fibronectin expressions using IF staining ( D ); collagen I content in the cell culture supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); and the protein level of Epac1, α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy OFs; # P < 0.05, ## P < 0.01 versus TAO OFs; && P < 0.01 versus healthy OFs + TGFβ1 + NC; $ P < 0.05, $$ P < 0.01 versus TAO OFs + TGFβ1 + NC. TAO, thyroid-associated orbitopathy; sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: OFs were subjected to 48 hours of treatment with 10 ng/mL TGFβ1 (MCE, Monmouth Junction, NJ, USA) for inducing activation.

Techniques: Over Expression, Transfection, Plasmid Preparation, CCK-8 Assay, Migration, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 knockdown amplifies the effects of TGFβ1 on normal and TAO OFs. ( A , B ) Healthy or TAO OFs were transfected with plasmids containing short-hairpin RNA targeting Epac1 (sh- Epac1 #1/#2), treated with TGFβ1 (10 ng/mL), and examined for Epac1 protein levels using immunoblotting; and ( B ) cell viability using CCK-8; sh- Epac1 #1 was selected for the following experiments. ( C – H ) Healthy or TAO OFs were transfected with sh- Epac1 #1, treated with TGFβ1 (10 ng/mL), and examined for cell migration using scratch wound healing and Transwell assays ( C , D ); relative expression of α-SMA and fibronectin using IF staining ( E ); the content of collagen I in cell culture supernatant using ELISA (F); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( G ); and the protein level of Epac1, α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( H ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. * P < 0.05, ** P < 0.01 versus healthy OFs + TGFβ1 + sh-NC; # P < 0.05, ## P < 0.01 versus TAO OFs + TGFβ1 + sh-NC. TAO, thyroid-associated orbitopathy; sh, short hairpin RNA; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: OFs were subjected to 48 hours of treatment with 10 ng/mL TGFβ1 (MCE, Monmouth Junction, NJ, USA) for inducing activation.

Techniques: Knockdown, Transfection, shRNA, Western Blot, CCK-8 Assay, Migration, Expressing, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 affects the STAT3 signaling. ( A ) A schematic diagram of Epac1’s role in the JAK/STAT3 signaling pathway during the OF activation process. ( B ) TAO OFs transfected with sh- Epac1 or Epacl overexpression vector, treated with TGFβ1, and determined for the protein level of p-STAT3 and STAT3 using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus NC-transfected OFs; ## P < 0.01 versus sh-NC-transfected OFs. ( C ) The protein level of p-STAT3 and STAT3 in TAO mouse OAT and OMT was determined using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy mice; # P < 0.05, ## P < 0.01 versus TAO + AAV-NC mice. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; sh, short-hairpin RNA; NC, negative control.

Article Snippet: OFs were subjected to 48 hours of treatment with 10 ng/mL TGFβ1 (MCE, Monmouth Junction, NJ, USA) for inducing activation.

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Virus, shRNA, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: STAT3 mediates the effects of Epac1 on TGFβ1-treated TAO OFs. ( A – G ) Under TGFβ1 treatment, TAO OFs were transfected with sh- Epac1 with or without the JAK-STAT pathway inhibitor Stattic (2 g/mL for 24 hours) and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); α-SMA and fibronectin expressions using IF staining ( D ); the content of collagen I in cell supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); the protein level of α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus TGFβ1 + sh-NC; ## P < 0.01 versus TGFβ1 + sh- Epac1 + Stattic. sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: OFs were subjected to 48 hours of treatment with 10 ng/mL TGFβ1 (MCE, Monmouth Junction, NJ, USA) for inducing activation.

Techniques: Transfection, CCK-8 Assay, Migration, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Cell metabolism

Article Title: Aerobic Glycolysis Controls Myeloid-Derived Suppressor Cells and Tumor Immunity via a Specific CEBPB Isoform in Triple-Negative Breast Cancer

doi: 10.1016/j.cmet.2018.04.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAP Over Expression Lentivirus LAP fragment with CMV promoter was cloned from LAP expression plasmid (plasmid #12557, Addgene) using KOD Xtreme hot start DNA polymerase (EMD Millipore).

Techniques: Control, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Lactate Assay, Cell Isolation, Gene Expression, shRNA, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 positively regulates the Hh pathway at postreceptor level. a Luciferase activity of NIH3T3 Shh-Light II cells treated for 24 h with SAG and increasing amounts of Leu-SH or DTT as control. b , c Quantitative real-time PCR (qRT-PCR) ( b ) and representative immunoblotting ( c ) analyses of Gli1 expression in the NIH3T3 murine fibroblasts transduced with lentiviral vectors encoding either control shRNA (shCTRL) or ERAP1 shRNA (shERAP1#1 and shERAP1#2) and treated with SAG or DMSO for either 24 or 48 h. In c ERAP1 expression was also evaluated and actin was used as loading control. d , f qRT-PCR analysis of Hh target genes expression in Ptch −/− ( d ) and SuFu −/− MEFs ( f ) both treated with Leu-SH (30 μM) or DTT as control. e Representative model of the constitutive activation of Smo or Gli1 in Ptch −/− and SuFu −/− MEFs, respectively. g , h qRT-PCR analysis of Hh target genes expression in Ptch −/− ( g ) and SuFu −/− MEFs ( h ) transduced with shCTRL or shERAP1 constructs. Data in b , d , f , g , and h are normalized to endogenous GAPDH and HPRT controls and expressed as the fold change respect to the control sample value. All data represent the mean of three independent experiments. Mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001 calculated with two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transduction, shRNA, Activation Assay, Construct

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 activates Hh signaling by impairing βTrCP protein expression. a – f Representative immunoblotting analyses of the indicated proteins in MEFs transfected with increasing amounts of vector encoding ERAP1 ( a , d ) or treated for 24 h with Leu-SH at the indicated concentration ( b , e ), or SAG (200 nM) and Leu-SH (30 μM) ( c ), or DTT as control. In f MEFs were transduced with shCTRL or shERAP1 and transfected with a vector encoding ERAP1. Actin ( a – c , f ) and tubulin ( d , e ) were used as loading controls. g ERAP1, Gli1 and βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 in the presence of small interfering RNAs (siRNAs) to a non-relevant mRNA (siCTRL) or murine βTrCP mRNA (siβTrCP). h βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 and treated with cycloheximide (CHX, 100 µg/mL) at different time points. Densitometry analysis of actin-normalized βTrCP values of three independent experiments is shown (right panel). i , j Endogenous βTrCP was immunoprecipitated from MEFs expressing the indicated proteins and treated with MG132 (50 μM) for 4 h ( i ) or increasing doses of Leu-SH for 24 h ( j ), followed by immunoblotting with an anti-HA antibody to detect conjugated HA-Ub. Blots were both reprobed with a βTrCP antibody. Bottom ERAP1 and βTrCP protein levels in total cell lysate. Actin was used as loading control. k Immunoblotting (upper panel) and densitometric analysis (lower panel) of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs and treated after 24 h with increasing amount of Leu-SH for 24 h. l Immunoblotting analysis of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs transduced with shCTRL or shERAP1. Actin was used as loading control

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Concentration Assay, Transduction, Immunoprecipitation

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 promotes βTrCP ubiquitylation by interacting with USP47. a – d MEFs were transfected with ERAP1 and/or Flag-USP47. Interaction between USP47 and ERAP1 was detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. e MEFs transfected with ERAP1 were stained with anti-ERAP1 and anti-USP47 antibodies. Green and red, USP47 and ERAP1 expressing cells, respectively. Nuclei were counter stained with Hoechst (Blue). Magnification ×60; Bars: 5 μm. Representative images from three independent experiments. f βTrCP and Gli steady state in USP47 +/+ and USP47 −/− MEFs. g βTrCP protein level in USP47 +/+ , USP47 −/− and USP47 −/− Flag-USP47 transfected MEFs. h βTrCP half-life in USP47 +/+ vs. USP47 −/− MEFs treated with CHX (100 µg/mL) at the indicated times. i βTrCP protein levels in MEFs transfected with empty vector as control or Flag-USP47 and treated with CHX (100 µg/mL) at different time points. j Gli1 protein levels in Ptch −/− MEFs transfected with empty vector as control or Flag-USP47 and treated after 24 h with CHX (100 µg/mL) for different time points. In g – j densitometric analysis of βTrCP and Gli1 protein levels of three independent experiments are shown (right panels). k MEFs were transfected with HA-Ub and increasing amount of ERAP1 in the presence or absence of Flag-USP47. Endogenous βTrCP was immunoprecipitated with an anti-βTrCP antibody and the ubiquitylated forms were revealed with an anti-HA antibody (upper panel). The blot was reprobed with an anti-βTrCP antibody. Flag-USP47, ERAP1 and βTrCP total protein levels are shown (lower panel). l MEFs were transfected with Flag-USP47 and increasing amount of ERAP1. Interaction between Flag-USP47 and endogenous βTrCP was assessed by immunoprecipitation and immunoblotting with the indicated antibodies. Actin was used as loading control. m MEFs were transfected with Flag-USP47 and treated for 24 h with Leu-SH at the indicated concentration. Interaction between Flag-USP47 and endogenous βTrCP was detected as described in l . Densitometric analysis of the Flag-USP47/βTrCP binding ratio representative of three independent experiments is shown (right panel). n MEFs were transduced with shCTRL or shERAP1 and transfected with Flag-USP47. Interaction between Flag-USP47 and endogenous βTrCP was assessed as described in l

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Expressing, Plasmid Preparation, Concentration Assay, Binding Assay, Transduction

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 impairs Hh-dependent growth of cerebellar granule cell progenitors. a H&E and immunohistochemical staining of Ki67, ERAP1 and Gli1 in the outer EGL during mouse cerebellum development. Magnification ×40. Scale bars represent 50 μm. b , c GCPs were isolated from 4-day-old mice and treated with either SAG alone or in combination with increasing doses of Leu-SH for 24 h. BrdU uptake ( b ) and mRNA levels of Gli1 ( c ) are shown. d , i GCPs isolated from 4-day-old mice were infected with lentiviral particles encoding for shERAP1 ( d – f ) or ERAP1 ( g – i ) and the corresponding controls, respectively. The percentage of BrdU uptake ( d , g ), mRNA ( e , h ), and protein levels ( f , i ) of Gli1 are shown. Results in c , e , h were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. legend, and represent the mean of three independent experiments. In f and i, actin was used as loading control. Mean ± S.D. * P < 0.05; ** P < 0.01 determined with two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Immunohistochemical staining, Staining, Isolation, Infection

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 impinges Hh-dependent tumor cell growth in vitro. a – f Primary cell cultures from Math1-cre/Ptc C/C mice MBs were treated with different amounts of Leu-SH. a , b Cells were counted with trypan blue at the indicated time points to evaluate the growth rate of viable cells ( a ) and the percentage of cell death ( b ). c Cleaved Caspase-3 protein levels in cells treated with Leu-SH at the indicated concentration for 24 h. d – f Percentage of BrdU uptake ( d ) and Gli1 mRNA ( e ), and protein ( f ) expression in MB cells treated with Leu-SH at the indicated concentrations for 24 h. g MB Stem-Like Cells (MB-SLCs) from Math1-cre/Ptc C/C mice were treated with Leu-SH as in (a) and counted with trypan blue at the indicated time points. h MB-SLCs were dissociated and treated with the indicated concentrations of Leu-SH or DTT as control. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was evaluated. The self-renewal MB-SLCs capability is expressed as percentage of neurosphere-forming cells (right). Representative bright field images of tumor neurospheres after Leu-SH treatment are shown (left). Scale bar 100 µM. i , j mRNA and protein expression levels of Hh target genes of MB-SLCs treated with the indicated concentrations of Leu-SH for 24 h. Actin was used as loading control. Results in e , i were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. legend. All data are representative of three independent experiments. Mean ± S.D. * P < 0.05; ** P < 0.01 calculated using two-tailed Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: In Vitro, Concentration Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 inhibition impairs Hh-dependent tumor growth in vivo. a – f NSG mice were grafted with spontaneous primary MB from Math1-cre/Ptc C/C mice. Tumor masses (150 mm 3 ) were intratumorally injected with Leu-SH. a Tumor growth was monitored. b Representative flank allograft average volumes (lower panel) and quantification of tumor explants (upper panel). c , d Ki67, NeuN, and cleaved Caspase-3 (Cl.Casp-3) immunohistochemical stainings of allograft tumor samples. d Quantification of immunohistochemical stainings shown in c . Scale bar 100 μm. e mRNA and f protein expression levels of Hh targets from tumors assayed in b . g Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor in NSG mice after i.p. injection of Leu-SH. Scale bars, 500 μm and 200 μm (upper and lower panels, respectively). h Representative average volume of orthotopic tumor. i – j NSG mice were grafted with spontaneous primary MB from Math1-cre/Ptc C/C mice genetically silenced for ERAP1 expression. i Representative images of mice and the explanted tumor masses. j Quantification of the flank allograft average tumor volume. ERAP1 protein expression is shown below. In f , j , actin was used as loading control. k H&E and representative Masson’s trichrome staining of tumors. Scale bar 100 μm. l mRNA levels of the indicated Hh target genes. m Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor genetically interfered for ERAP1 before the injection in NSG mice cerebella. Scale bars, 500 and 200 μm (upper and lower panels, respectively). n – p ERAP1 accelerates Hh-MB formation. n Tumor volume of mice subcutaneously transplanted with GCPs from tumor-prone Math1-cre/Ptc C/C animals overexpressing ERAP1. o Representative flank allograft average volumes (lower panel) and quantification of the explanted tumor masses (upper panel). p mRNA expression of Hh target genes from the tumor masses assayed in o . q Survival curves of Math-cre/Ptc C/C mice treated with Leu-SH or vehicle. Results in e , l , p were normalized to endogenous GAPDH and HPRT controls and expressed as in Fig. . All data represent the mean of three independent experiments. Mean ± S.D. of tumor ( n = 6) for each treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 calculated by two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Inhibition, In Vivo, Injection, Immunohistochemical staining, Expressing, Derivative Assay, Staining

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: A representative model showing the role of ERAP1 in Hh-dependent tumorigenesis. ERAP1 promotes ubiquitylation and proteasomal degradation of βTrCP by sequestering USP47. This event leads to increase of Gli1 and Gli2 protein levels and decrease of Gli3R, thus triggering the Hh pathway and favoring cell growth and tumorigenesis. In the absence of ERAP1, USP47 binds and stabilizes βTrCP, which, in turn, promotes ubiquitylation and proteasomal degradation of Gli1 and Gli2, and ubiquitylation and proteolytic cleavage of Gli3 into the repressor form Gli3R. These events lead to the repression of the Hh pathway and inhibition of cell proliferation and tumor growth

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Inhibition

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 inhibited PI3K/AKT and Raf/MEK/ERK signaling pathways triggered by TGFβ1. (A) The total proteins extracted from cells were analyzed for the expression of AKT, ERK and Bax using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-Bax and anti-GAPDH. (B) ELISA analysis of TGFβ1 secretion from AsPC-1/vector and AsPC-1/TIPE2 cells. (C) Immunohistochemistry analysis of the TGFβ1 expression in AsPC-1/vector and AsPC-1/TIPE2 tumor tissues. (D) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in AsPC-1/vector and AsPC-1/TIPE2 cells. (E) AsPC-1 cells were seeded in 6-well plate and added with or without anti-TGFβ1 antibody or rhTGFβ1 protein. After 48 h incubation, the total proteins extracted from the cultured AsPC-1 cells were analyzed for the expression of AKT and ERK using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK and anti-GAPDH. Data shown were representative of three independent experiments. Values are presented as means ± SD. ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Incubation, Cell Culture

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 suppressed the growth of pancreatic cancer through inhibiting TGFβ1 expression in subcutaneous tumor model. (A) The tumor volume was measured weekly one week after tumor inoculation and the tumor growth curve was calculated. (B, C) The tumors were isolated, weighted and photographed five weeks after tumor cells inoculation. (D) ELISA analysis of TGFβ1 secretion from Panc02/vector and Panc02/TIPE2 cells. (E) Immunohistochemistry analysis of the TGFβ1 expression in Panc02/vector and Panc02/TIPE2 tumor tissues. (F) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in Panc02/vector and Panc02/TIPE2 cells. The data shown are the representative of three experiments. Values are presented as means ± SD. *p < 0.05, ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Western Blot

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A T98G/Cas9/CD4/CXCR4/Firefly cells were pre‐treated with IFN for 24 h prior to infection with increasing doses of NL4‐3 Renilla (NL4‐3/Nef‐IRES‐Renilla) (indicated in ng p24 Gag ). Renilla activity was normalized to Firefly activity and the relative infection efficiencies are shown. Data represent the average and standard deviation (s.d.) of 3 biological replicates. B GeCKO control cells and enriched cells from 3 successive rounds of selection (Round 1, 2, and 3, as indicated) were treated with IFN or not and infected with GFP‐expressing lentiviral vectors. The percentage of infected cells was evaluated by flow cytometry 2 days post‐infection. Data represent the average of 2 biological replicates. C The indicated primary cells or immortalized cell cultures were treated with IFN for 24 h or left untreated. RNA was subsequently extracted and DDX42 and ISG15 (a prototype ISG) mRNA levels were quantified by RT‐qPCR; ActinB and GAPDH were used as endogenous controls. The bar chart shows the relative levels of expression of DDX42 and ISG15 in the presence or absence of IFN treatment. Data represent the mean ± S.E.M of 3 biological replicates.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Infection, Activity Assay, Standard Deviation, Control, Selection, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A Screen strategy. GeCKO cell populations (obtained by transduction of T98G/Cas9 cells with GeCKO v2 LV library) were IFN‐treated, challenged with HIV‐1 LVs coding for an antibiotic resistance gene and selected. Three rounds of IFN treatment, infection and selection were performed. Genomic DNAs of initial GeCKO and 3‐time selected populations were extracted, the sgRNA‐coding sequences amplified and sequenced. B Candidate gene identification. MAGeCK computational statistical tool (Li et al , ) was used to establish a Robust Rank Aggregation (RRA) score for each gene based on sgRNA enrichment and number of sgRNAs per gene. Genes belonging to the type 1 IFN response pathway (in blue) and DDX42 (in red) are shown (respective ranks into brackets) for 2 independent screens (the results of which were merged in the analysis). The dashed line indicates the significance threshold. C Candidate validation. T98G/Cas9/CD4/CXCR4/Firefly KO populations were generated for the 25 top hits of each screen. The control (CTRL) condition represents the mean of 4 negative CTRL populations, obtained with 4 non‐targeting sgRNAs; IFNAR1 and MX2 KO populations were used as positive controls. KO cell populations were treated with IFN and infected with HIV‐1 Renilla (NL4‐3/Nef‐IRES‐Renilla) and luciferase signals were measured 30 h later (Renilla signals were normalized to Firefly). IFN inhibition (i.e. ratio of untreated / IFN‐treated conditions) was calculated and set at 100% inhibition for CTRL. Data from technical duplicates are shown.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Transduction, Infection, Selection, Amplification, Biomarker Discovery, Generated, Control, Luciferase, Inhibition

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A Top: DDX42 KO and CTRL KO U87‐MG/CD4/CXCR4/Cas9/Firefly cells were generated using 3 sgRNAs and 4 non‐targeting sgRNAs, respectively (for CTRL, the average of data obtained with 4 cell populations is shown). Cells were treated or not with IFN 24 h prior to infection with HIV‐1 Renilla (NL4‐3/Nef‐IRES‐Renilla). Relative luminescence results for IFN‐treated and ‐untreated conditions are shown. Two‐way ANOVA on log‐transformed data with Sidak's test. Bottom: Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42‐depleted populations; Actin served as a loading control. B siRNA‐transfected U87‐MG/CD4/CXCR4 cells were treated or not with IFN for 24 h prior to infection with HIV‐1 Renilla. Relative luminescence results for IFN‐treated and ‐untreated conditions are shown. Multiple linear regression analysis. C DDX42 silencing efficiency measured by RT‐qPCR (top) and immunoblot (bottom) in parallel samples from B. D DDX42‐depleted cells were infected with HIV‐1 (WT NL4‐3), and infection efficiency was measured by CA p24 Gag intracellular staining and flow cytometry analysis. When indicated, cells were treated with azidothymidine (AZT) and lamivudine (3TC). Two‐way ANOVA on log‐transformed data with Dunnett's test. E CTRL and IRF9/STAT1 KO cells were pre‐treated or not with IFN for 24 h and infected with HIV‐1 Renilla (NL4‐3/Nef‐IRES‐Renilla). Infection efficiency was assessed 24 h later by measuring Renilla activity. Two‐way ANOVA on log‐transformed data with Dunnett's test. F siRNA‐transfected MDMs were infected with a CCR5‐tropic version of HIV‐1 Renilla (NL4‐3/R5/Nef‐IRES‐Renilla). Relative luminescence results from biological triplicates performed with cells from different donors are shown. Two‐way ANOVA on log‐transformed data with Dunnett's test. G Primary CD4 + T cells were electroporated with Cas9‐sgRNA RNPs using 2 non‐targeting sgRNAs (sgCTRL1 and 2) and 5 sgRNAs targeting DDX42. Top: Cells were then infected with HIV‐1 Renilla (NL4‐3/Nef‐IRES‐Renilla) and relative infection efficiencies obtained with cells from 3 donors are shown. Two‐way ANOVA on log‐transformed data with Dunnett's test. Bottom: DDX42 protein levels were determined by immunoblot, Actin served as a loading control. A representative immunoblot is shown. H Firefly‐ or DDX42‐expresssing U87‐MG/CD4/CXCR4 cells were infected with HIV‐1 Renilla (NL4‐3/Nef‐IRES‐Renilla). Relative infection efficiencies are shown. Multiple linear regression analysis. Data information: (A–D) and (F–H), Data represent the mean ± S.E.M of 3 biological replicates, (E) Data represent the mean ± S.E.M of 4 biological replicates. P values are denoted as follow: ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Generated, Infection, Transformation Assay, Western Blot, Control, Transfection, Quantitative RT-PCR, Staining, Flow Cytometry, Activity Assay

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A Cell viability of siRNA‐transfected U87‐MG/CD4/CXCR4 cells was assessed 72 h post‐transfection by measuring ATP levels. Data represent the mean ± S.E.M of 3 biological replicates. B U87‐MG/CD4/CXR4 cells were transfected with siRNAs, pre‐treated or not with IFN and infected with WT HIV‐1 (NL4‐3) in the presence or not of reverse transcription inhibitors (AZT/3TC). 48 h later, cells were lysed, RNA extracted and RT‐qPCR analysis performed to measure HIV‐1 RNAs. Data represent the mean ± S.E.M of 4 biological replicates. Two‐way ANOVA on log‐transformed data with Sidak's test. C Supernatants from (B) were harvested 48 h post‐infection and AlphaLisa used to measure CA p24 Gag production. Data represent the mean ± S.E.M of 4 biological replicates. Two‐tailed, unpaired t test. D RT‐qPCR analysis was performed RNA samples from (B) to measure relative expression of the ISG OAS1, ISG15, and MX1 (normalized to actin and GAPDH). Data represent the mean ± S.E.M of 4 biological replicates. Two‐way ANOVA with Dunnett's test. E U87‐MG/CD4/CXCR4 cells were transduced to express Cas9 and sgRNAs either targeting nothing (CTRL) or IRF9 and STAT1 (IRF9/STAT1). After 2 weeks, cells were treated or not with IFN, and, 48 h later, IFITM3 and MX2 induction was analyzed by immunoblotting, Actin served as a loading control. A representative immunoblot is shown. F siRNA‐transfected MDMs were harvested 48 h post‐transfection for RNA extraction and quantification of DDX42 mRNA levels by RT‐qPCR. Actin and GAPDH were used as endogenous controls. Data represent the mean ± S.E.M of 3 biological replicates performed with cells from different blood donors (parallel samples from Fig ). One‐way ANOVA with Dunnett's test. G U87‐MG/CD4/CXCR4 cells were transduced with lentiviral vectors expressing either Firefly (negative control), WT DDX42 (WT) or a motif I point mutant, which has an impaired ATPase activity (K303E). Transduced cells were infected with HIV‐1 Renilla (NL4‐3/Nef‐IRES‐Renilla) and the infection efficiency was assessed 24 h later by measuring Renilla activity. Data represent the mean ± S.E.M of 3 biological replicates. Two‐way ANOVA on log‐transformed data with Dunnett's test. Data information: P values are denoted as follow: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available online for this figure.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Transfection, Infection, Reverse Transcription, Quantitative RT-PCR, Transformation Assay, Two Tailed Test, Expressing, Western Blot, Control, RNA Extraction, Transduction, Negative Control, Mutagenesis, Activity Assay

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A siRNA‐transfected U87‐MG/CD4/CXCR4 cells were infected with WT HIV‐1 (NL4‐3) and relative amounts of Minus Strand Strong Stop (MSSS), 1 st and 2 nd Strand Transfer DNAs, and nuclear forms of HIV‐1 DNA (proviral DNA, and 2‐LTR circles) were quantified by qPCR. DNAs from cells infected for 48 h in the presence of AZT and 3TC were used as a control. Mixed‐effects analysis on log‐transformed data with Dunnett's test. B Silencing efficiency in parallel samples from A. C Heat‐annealed ODN/RNA 1–294 complex was incubated with HIV‐1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analyzed by PAGE 8% (P/T: primer/template; SSDNA: Strong‐Stop DNA). A representative autoradiograph is shown. D Quantification of 3 biological replicates performed as in C. Two‐way ANOVA on log‐transformed data with Dunnett's test. E PLAs were performed in MDMs infected with HIV‐1 or not (N.I. CTRL), using anti‐Capsid and anti‐DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale‐bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD ( n > 65 cells per condition). Mann–Whitney test. F siRNA‐transfected TZM‐bl cells were infected with the indicated VSV‐G‐pseudotyped, replication competent viruses and β‐galactosidase signals measured 24 h later. Two‐way ANOVA on log‐transformed data with Dunnett's test. G Silencing efficiency in parallel samples from (F). H siRNA‐transfected U87‐MG/CD4/CXCR4 cells were infected with HIV‐1‐ HIV‐2‐ FIV‐ EIAV‐based, GFP‐coding LVs and infection efficiency was scored 24 h later by measuring the percentage of GFP expressing cells by flow cytometry. Two‐way ANOVA on log‐transformed data with Dunnett's test. I siRNA‐transfected U87‐MG/CD4/CXCR4 cells were infected with GFP‐coding B‐MLV retroviral vector and infection efficiency measured 24 h later by flow cytometry. Simple linear regression analysis. J HEK293T were co‐transfected with GFP‐coding LINE‐1 plasmids (RPS‐GFP or LRE3‐GFP) or with an inactive LINE‐1 plasmid (JM111) together with either a Firefly‐ or DDX42‐coding plasmid. GFP expression was measured by flow cytometry 7 days later. Two‐way ANOVA on log‐transformed data with Sidak's test. K HEK293T were co‐transfected with pRPS‐GFP and a Flag‐Firefly‐ (negative control) or Flag‐DDX42‐coding plasmid, followed by Flag immunoprecipitation and immunoblot analysis. A representative immunoblot is shown. L Left, RNA extraction and LINE‐1 RT‐qPCR on parallel samples from (K). Two‐way ANOVA on log‐transformed data with Sidak's test. Right, Percentage of immunoprecipitated RNA. T ‐test on log‐transformed data. Data information: (A, B) and (D–J), Data represent the mean ± S.E.M of 3 biological replicates, (L), Data represent the mean ± S.E.M of 5 biological replicates. P values are denoted as follow: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Transfection, Infection, Control, Transformation Assay, Incubation, Recombinant, Reverse Transcription, Autoradiography, Staining, Microscopy, MANN-WHITNEY, Expressing, Flow Cytometry, Retroviral, Plasmid Preparation, Negative Control, Immunoprecipitation, Western Blot, RNA Extraction, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A DDX42‐depleted U87‐MG/CD4/CXCR4 cells were infected with the indicated amounts of NL4‐3/Nef‐IRES‐Renilla viruses carrying the β‐lactamase (BlaM)‐Vpr fusion protein for 3 h. The cells were subsequently loaded with CCF2‐AM substrate dye for 2 h, washed extensively and incubated for another 16 h for the reaction to develop. Cells positive for the CCF2‐AM product were scored by flow cytometry. Data represent the mean ± S.E.M of 3 biological replicates. Two‐way ANOVA on log‐transformed data with Dunnett's test. P values are denoted as follow: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. B Elution profile of His‐DDX42. Top right, SDS‐PAGE followed by Coomassie coloration analysis of the pooled fractions. C Heat‐annealed ODN/RNA 1–294 complex was incubated with HIV‐1 RT and the indicated, increasing concentrations of recombinant DDX42 (Fig ). Reverse transcription was initiated by addition of dTTP, dGTP, dCTP and ddATP. Extension was for 1, 5, 20 or 60 min and samples were analyzed by PAGE 12% (P/T: primer/template; +5: extension of 5 nucleotides). A representative autoradiograph is shown. D Quantified data correspond to the ratio of extension products / unextended P/T at different time points. Data represent the mean ± S.E.M of 4 biological replicates. Two‐way ANOVA on log‐transformed data with Dunnett's test. P values are denoted as follow: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. E MDMs were fixed, endogenous DDX42 and the nuclei were visualized using DDX42‐specific antibodies and Hoechst staining, respectively, and confocal microscopy. Representative images are shown. Scale bar, 10 μm.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Infection, Incubation, Flow Cytometry, Transformation Assay, SDS Page, Recombinant, Reverse Transcription, Autoradiography, Staining, Confocal Microscopy

Journal: EMBO Reports

Article Title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses

doi: 10.15252/embr.202154061

Figure Lengend Snippet: A Relative IAV‐Nanoluciferase (IAV‐NLuc) infection efficiency in siRNA‐transfected U87‐MG cells (Nanoluciferase activity 16 h post infection). Multiple linear regression analysis. B Relative VSV‐Firefly infection efficiency in siRNA‐transfected U87‐MG cells (Firefly activity 24 h post infection). Multiple linear regression analysis. C Relative MV‐GFP infection efficiency in siRNA‐transfected Huh‐7 cells (GFP+ cells scored 24 h post infection). Multiple linear regression analysis. D Relative ZIKV‐Nanoluciferase infection efficiency in siRNA‐transfected U87‐MG cells (Nanoluciferase activity 24 h post infection). Multiple linear regression analysis. E Relative CHIKV‐Nanoluciferase infection efficiency in siRNA‐transfected U87‐MG cells (Nanoluciferase activity 24 h post infection). Multiple linear regression analysis. F WT CHIKV infection efficiency in control (−) and IFN‐treated (+), siRNA‐transfected U87‐MG cells analyzed by RT‐qPCR on genomic RNA. Two‐way ANOVA on log‐transformed data with Dunnett's test. G Viral production in cell supernatants from U87‐MG cells with WT CHIKV (24 h post‐infection, MOI 1) measured by plaque assays. Two‐way ANOVA on log‐transformed data with Dunnett's test. H CTRL and IRF9/STAT1 KO U87‐MG/CD4/CXCR4 cells were pre‐treated or not with IFN for 24 h and infected with CHIKV‐NLuc. Infection efficiency was assessed 24 h later by measuring Nanoluciferase activity. Two‐way ANOVA on log‐transformed data with Dunnett's test. I Relative WT SARS‐CoV‐2 infection in siRNA‐transfected A549‐ACE2 cells (RdRp RT‐qPCR 48 h post‐infection). Mixed‐effects analysis on log‐transformed data with Dunnett's test. J Viral production in A549‐ACE2 cell supernatants from I (48 h post‐infection, MOI 0.05) measured by plaque assays. Two‐way ANOVA on log‐transformed data with Dunnett's test. K Relative HCoV‐229E‐Renilla infection efficiency in siRNA‐transfected Huh7.5.1 cells (Renilla activity 24 h post infection). Two‐way ANOVA on log‐transformed data with Dunnett's test. L CTRL and IRF9/STAT1 KO A549‐ACE2 cells were pre‐treated or not with IFN for 24 h and infected with SARS‐CoV‐2‐Nanoluciferase. Infection efficiency was assessed 24 h later by measuring Nanoluciferase activity. Two‐way ANOVA on log‐transformed data with Dunnett's test. Data information: Data represent the mean ± S.E.M. of 3 biological replicates (A‐E, F for MOI 0.5, and K) or 4 biological replicates (F for MOI 0.1, G–J and L). P values are denoted as follow: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cell surface staining with anti‐CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers.

Techniques: Infection, Transfection, Activity Assay, Control, Quantitative RT-PCR, Transformation Assay

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A induces proliferation of human non-small-cell lung cancer (NSCLC) cells harboring wild-type (WT) or mutant epidermal growth factor receptor (EGFR). ( A ) Western blot analysis of IL-17A expressions in PC9, HCC827, and A549 cells after transfecting a control vector (Neo) or IL-17A-expressing vector (IL-17A-His). ( B ) Dot blot analysis of secreted IL-17A using conditioned media of IL-17A-His-transfected PC9, HCC 827, and A549 cells. The uncropped blots are shown in the . ( C ) Proliferation rates of IL-17A-overexpressing PC9, HCC827, and A549 cells were measured by performing a CCK8 assay. Proliferation rates had significantly increased in IL-17A-His-transfected NSCLC cells after 24 (left) and 48 h (right) compared to control cells. ( D ) PC9, HCC827, and A549 cells were treated with 0, 1, 10, or 50 ng/mL recombinant human (rh)IL-17A for 24 and 48 h. The effect of rhIL-17A on proliferation of these NSCLC cells was evaluated by a CCK-8 assay. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control group.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Mutagenesis, Western Blot, Control, Plasmid Preparation, Expressing, Dot Blot, Transfection, CCK-8 Assay, Recombinant

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A facilitates phosphorylation of epidermal growth factor receptor (EGFR) and Met and subsequently induces EGFR-tyrosine kinase inhibitor (TKI) resistance in human non-small-cell lung cancer (NSCLC) cells harboring mutant-EGFR. ( A , B ) Detection of phosphorylated (p)-EGFR and its downstream signaling by Western blotting after overexpression of IL-17A ( A ) and treatment with rhIL-17A at different concentrations and time points ( B ) in EGFR-mutant PC9 cells. ( C ) Western blot analysis of p-EGFR in PC9 cells after transducing shIL-17RC or control shRNA (shLuc) and treatment with rhIL-17A or the vehicle. Quantitative results of p-EGFR proteins were adjusted to GAPDH protein levels. ( D ) A CCK8 assay was performed to evaluate the proliferation inhibitory effect of afatinib treatment for 72 h in PC9 or HCC827 cells with or without IL-17A overexpression *** p < 0.001, ## p < 0.01, ### p < 0.001. ( E ) Proliferation rates of PC9 cells that received different concentrations of afatinib combined with or without rhIL-17A for 24 h. *** p < 0.001 compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the afatinib-treated only group. ( F ) Differential expression levels of phosphorylated receptor tyrosine kinases (RTKs) in PC9 cell lysates following 24 h treatment with rhIL-17A. An antibody array (R&D Systems) was used to detect 49 different phosphorylated human RTKs. The left panel shows representative array blots. The right panel shows a quantitative analysis of phosphorylated RTKs using a densitometer. Values are presented as the mean ± SD. n = 2. ( G , H ) Detection of phosphorylated (p)-Met by Western blotting after overexpression of IL-17A ( G ) and treatment with rhIL-17A at different concentrations and time points ( H ) in PC9 cells. ( I ) Detection of p-Met by Western blotting in PC9/IL-17A and PC9/Neo cells receiving different concentrations of SU11274. The uncropped blots are shown in the . ( J ) Proliferation rates of rhIL-17A-treated or vehicle-treated PC9 cells after receiving afatinib or afatinib+SU11274 for 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control group. ### p < 0.001 compared to the afatinib-treated only group.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Over Expression, Control, shRNA, CCK-8 Assay, Quantitative Proteomics, Ab Array

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A synergizes with epidermal growth factor (EGF) to trigger EGF receptor (EGFR) activation via preventing EGF-mediated EGFR degradation in human non-small-cell lung cancer (NSCLC) cells harboring wild-type (WT)-EGFR. ( A , B ) Differential expression levels of phosphorylated kinases in A549 cell lysates following 10 min treatment with EGF or EGF + rhIL-17A. An antibody array (R&D Systems) was used to detect 37 different phosphorylated kinases and two related total proteins. Representative array blots are shown in ( A ). Quantitative analysis of phosphorylated kinases or total proteins using a densitometer are shown in ( B ). ( C ) Detection of phosphorylated (p)-EGFR and total EGFR by Western blotting after treatment of A549 cells with EGF or EGF+rhIL-17A for the indicated time points. ( D ) Western blot analysis of p-EGFR and total EGFR in A549 cells after transducing shIL-17RC or control shRNA (shLuc) and treatment with EGF or EGF+rhIL-17A for 10 min. ( E ) IL-17A-His-transfected A549 or control cells were treated with EGF (100 ng/mL) for the indicated time points, and expression levels of p-EGFR and total EGFR were further evaluated by a Western blot analysis. ( F ) A549/shLuc and A549/shIL-17RC cells were transfected with IL-17A-His or a control vector and treated with EGF for 10 min, and total EGFR was further detected. Quantitative results of p-EGFR and total EGFR proteins from ( C – F ) were, respectively, adjusted to total EGFR and β-actin or GAPDH protein levels. The uncropped blots are shown in the . ( G ) A CCK8 assay was performed to evaluate proliferation rates of A549 cells which were treated with the vehicle, EGF (100 ng/mL), rhIL-17A (10 ng/mL), or EGF+rhIL-17A for 24 h. * p < 0.05, compared to the EGF- or rhIL-17A-treated only group.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Activation Assay, Quantitative Proteomics, Ab Array, Western Blot, Control, shRNA, Transfection, Expressing, Plasmid Preparation, CCK-8 Assay

Journal: Cancers

Article Title: Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

doi: 10.3390/cancers15133288

Figure Lengend Snippet: Interleukin (IL)-17A prevents epidermal growth factor (EGF)-mediated EGF receptor (EGFR) degradation via impairing the transport of EGFR to lysosomes in human non-small-cell lung cancer (NSCLC) cells harboring wild-type (WT)-EGFR. ( A , B ) Serum-starved A549 cells were pretreated with MG132 (20 µM) ( A ) or bafilomycin A1 (20 nM) ( B ) for 1 h, and then 100 ng/mL EGF was added for the indicated time points. Cell lysates from ( A , B ) were subjected to a Western blot analysis to detect phosphorylated (p)-EGFR and total EGFR expressions. The uncropped blots are shown in the . ( C ) Serum-starved A549 cells were treated with EGF (100 ng/mL) alone or EGF combined with rhIL-17A (10 ng/mL) for the indicated time points (10 and 30 min). Membrane EGFR levels were detected by a FASC analysis after staining with a mouse mAb to EGFR or mouse isotype control antibodies. The mean fluorescence intensity was a measure of the level of EGFR expression. *** p < 0.001 compared to the control group. ( D ) Different colocalization of lysosomal-associated membrane protein 1 (LAMP1) and endocytosed EGFR after 30 min of EGF or EGF+rhIL-17A treatment. Left panel: Cells were fixed, permeabilized, and stained with anti-EGFR (green), anti-LAMP1 (red), and DAPI (blue) for nuclear staining, and further examined by confocal microscopy. White arrows point to colocalization of EGFR and lysosomes. Orange arrows point to the nuclear translocation of EGFR. Right panel: Colocalization rate of EGFR and lysosome or nucleus was analyzed by MetaMorph software (version 7.8.4.0). * p < 0.05, ** p < 0.01 compared to the vehicle control group. # p < 0.05 compared to the EGF-treated alone group. ns: No Significance.

Article Snippet: Santa Cruz Biotechnology (Santa Cruz, CA, USA) provided antibodies for IL-17A (sc-374218), EGFR (sc-101), lysosomal-associated membrane protein 1 (LAMP1; sc-20011), and GAPDH (sc-32233), while Proteintech Group (Chicago, IL, USA) supplied the antibody for His-tag (66005-1-ig).

Techniques: Western Blot, Membrane, Staining, Control, Fluorescence, Expressing, Confocal Microscopy, Translocation Assay, Software

Journal: International Journal of Molecular Medicine

Article Title: MicroRNA-218 inhibits the malignant phenotypes of glioma by modulating the TNC/AKT/AP-1/TGFβ1 feedback signaling loop

doi: 10.3892/ijmm.2021.5038

Figure Lengend Snippet: Blockade of the TNC/AKT/AP-1/TGFβ1-positive feedback loop by miR-218 (A) The effect of miR-218 mimics on the expression or phosphorylation of the indicated proteins in U251 and SHG44 cells was assessed by western blot analysis β-actin was used as a loading control (B) Dual-luciferase reporter system was used to assess the effect of miR-218 mimics on AP-1 activity in U251 and SHG44 cells Empty vector was used as the control The ratio of the Luc/ Renilla activity was presented as the mean ± SD of three independent assays (C) Lentivirus-encoding miR-218 and control lentivirus were transfected into cells to establish tumor xenografts The effect of miR-218 on the expression or phosphorylation of the indicated proteins in the xenograft tumors was evaluated by western blot analysis (D) U251 and SHG44 cells transfected with miR-218 or NC mimics were treated with or without exogenous TGFβ1 Western blot analysis was performed to detect the expression or phosphorylation of the indicated proteins β-actin was used as a loading control (E) Dual-luciferase reporter system was performed to assess AP-1 activity Empty vector was used as the control The ratio of the Luc/ Renilla activity was expressed as the mean ± SD (F) A schematic model illustrating the mechanism of miR-218 inhibiting malignant phenotypes of glioma cells * P<005 and ** P<001 miR, microRNA; TNC, tenascin C; TGF β1, transforming growth factor β1; AP-1, activator protein 1; Luc, luciferase; NC, negative control

Article Snippet: The human glioma cell lines, U251 and SHG44, were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China) Cells were maintained in DMEM (Invitrogen; Thermo Fisher Scientific, Inc) supplemented with 10% FBS (Biological Industries), 100 IU/ml penicillin and 100 µ g/ml streptomycin, at 37°C in a humidified atmosphere with 5% CO 2 All cell lines used in this study were authenticated by short tandem repeat (STR) analysis using the Cell ID System (Promega Corporation) in March 2019 with maximum 20 passages before the cells were analyzed Meanwhile, the one-step Quickcolor Mycoplasma Detection kit (Shanghai Yise Medical Technology Co, Ltd; http://www.yisemedcom ) was used according to the manufacturer's instructions, to demonstrate that these cell lines were not contaminated by mycoplasma In some experiments, cells were treated with recombinant human TGFβ1 proteins (10 ng/ml; Sino Biological, Inc) for 24 h The control group had the same volume of a vehicle

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Negative Control

Journal: Nature communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole.

doi: 10.1038/ncomms12567

Figure Lengend Snippet: Figure 3 | Cep295 is essential for the ability of a new mother centriole to recruit pericentriolar material and to generate a procentriole. (a) Schematic of Cep295 function described in this figure. (b) Three colour staining of centrioles in control and Cep295-depleted cells. The cells were stained with the indicated antibodies. ODF2 was used as an older mother centriole marker. Arrows point to a new mother centriole (M: older mother centriole; NM: new mother centriole). Scale bar, 1 mm. (c,d) HeLa cells transfected with control siRNA or Cep295 siRNA for 72 h were stained with indicated antibodies. CP110 and centrin were used as centriole markers48. Scale bar, 1 mm. (c) The number shown in the bottom panels represents the number of Plk4/HsSAS-6/STIL foci. (d) Histograms represent frequency of interphase cells with ^2 Plk4/HsSAS-6/STIL/CPAP foci in each condition. Values are mean percentages±s.e.m from three independent experiments (N ¼ 30 for each condition). ***Po0.001; **Po0.01, (two-tailed t-test). (e) HeLa cells were treated with control siRNA or Cep295 siRNA, followed by transfection with an empty vector ( ) or, pCMV5-Plk4DPEST-FLAG wild-type. The cells were stained with the indicated antibodies. An arrow points to the defect in multiple centriole formation induced by PLK4 over-expression, upon Cep295 depletion. Scale bars, 5 mm in the low-magnified view, 1 mm in the inset. Schematic illustrations show frequency of interphase cells with 2 or 1 over-duplicated centriole foci. (f) Cep295-depleted HeLa cells were stained with the indicated antibodies. Almost all control cells harbour Z2 Cep192 and Z2 Cep152 foci per cell, whereas only 12 and 5% of Cep295-depleted cells have Z2 Cep192 and Z2 Cep152 foci per cell, respectively. Scale bar, 1 mm. (g,h) To monitor the expression levels of Cep295 at the mother and daughter centrioles, the triple staining analysis was performed with the indicated antibodies as shown in the representative panels. HeLa cells were transfected with control or Cep295 siRNA for 24 h. (g) Arrows point to the defective recruitment of PCM components in the Cep295-depleted cells just after disengagement. Scale bar, 1 mm. (h) Histograms represent frequency of late mitotic cells with the indicated category in each condition. Values are mean percentages±s.e.m from three independent experiments (N ¼ 30 for each condition). **Po0.01 (two-tailed t-test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), g-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and a-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Staining, Control, Marker, Transfection, Two Tailed Test, Plasmid Preparation, Over Expression, Expressing