Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Molecular Weight, Marker

Journal: Cell Death and Differentiation

Article Title: Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration

doi: 10.1038/s41418-022-00967-4

Figure Lengend Snippet: Key reagents and resources used in this study.

Article Snippet: TBK1/NAK (D1B4) Rabbit mAb , Cell Signaling Technology , cat#3504.

Techniques: Marker, Recombinant, Transfection, Drug discovery, Gene Expression, Microarray, Software

Journal: Cell stem cell

Article Title: A M etformin -R esponsive M etabolic P athway controls distinct steps in gastric progenitor fate decisions and maturation

doi: 10.1016/j.stem.2020.03.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Vectastain Elite ABC HRP Kit , Vector Laboratories , Cat#PK6100.

Techniques: Recombinant, SYBR Green Assay, Protein Extraction, Plasmid Preparation, Microarray, Expressing, Software, Imaging

Journal: The Journal of Experimental Medicine

Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

doi: 10.1084/jem.20140280

Figure Lengend Snippet: The response to type I IFNs in TYK2-deficient cells is impaired but not abolished. (A–D) Western blot of proteins extracted from EBV–B cells from a healthy control (C), TYK2-deficient patients (P1, P2, P5, P7, and P8), and a STAT1-deficient patient (STAT1*), with and without IFN-α (10 5 IU/ml) or IFN-β (3.2 × 10 4 IU/ml) stimulation for 30 min. (A and B) Abs against phosphorylated STAT1 (pSTAT1), STAT1, and tubulin were used. After analysis by densitometry, a p-value <0.01 (0.0018) for the two-tailed Student’s t test for the comparison of IFN-α–pSTAT1 controls and TYK2-deficient patients (TYK2) was found. (C and D) Abs against phosphorylated STAT3 (pSTAT3), STAT3, and tubulin were used. After analysis by densitometry, a p-value <0.01 (0.0008) for the two-tailed Student’s t test for the comparison of IFN-α–pSTAT3 controls and TYK2-deficient patients (TYK2) was found. (E) Western blot detecting pSTAT1 and pSTAT3 in mock-transduced (left) or TYK2-transduced (right) EBV–B cells from a healthy control and P1, P2, P3, and P5, without (−) and with (+) stimulation for 30 min with 10 5 IU/ml IFN-α. (F and G) EMSA performed on EBV–B cells from a healthy control (C1), three TYK2-deficient patients (P1, P2, and P3), a patient with complete STAT1 deficiency (STAT1*), with (+) and without (−) stimulation with IFN-α (10 5 IU/ml) or IFN-β (3.2 × 10 4 IU/ml) for 30 min, with a GAS probe (F) or an ISRE probe (G). (H) EMSA performed on SV40-fibroblasts from healthy controls (C1 and C2), the TYK2-deficient patient P2 (P2), and a patient with complete STAT1 deficiency (STAT1*), with (+) and without (−) stimulation with 10 5 IU/ml IFN-α or 3.2 × 10 4 IU/ml IFN-β for 30 min, probed for GAS (top) and ISRE (bottom). (I) Induction of ISG15 (left) and MX1 (right) relative to GUS , as assessed by RT-qPCR on mRNA extracted from EBV–B cells from three healthy controls (mean C), TYK2-deficient patients (P1 and P2), and a patient with complete STAT1 deficiency (STAT1*), without (−) or with stimulation for 6 h with 3.2 × 10 4 IU/ml IFN-β. (J) EBV–B cells from two healthy controls (C1 and C2), two TYK2-deficient patients (P1 and P2), and a patient with complete STAT1 deficiency (STAT1*) were left untreated (−) or were treated with 10 5 IU/ml IFN-α 18 h before infection with VSV (MOI = 1). Viral load was determined at 48 h and is represented as log 10 TCID50/ml. (K) SV40-fibroblasts from two healthy controls (C1 and C2), TYK2-deficient patient P2 (P2), and a STAT1-deficient (STAT1*) patient were or were not (−) treated with 10 5 IU/ml IFN-α 18 h before infection with VSV (MOI = 10) for 6, 8, or 24 h. Viral load was determined at these three time points. (L) HVS–T cells from two healthy controls (C1 and C2), the TYK2-deficient patient P2 (P2), and a STAT1-deficient (STAT1*) patient were or were not (−) treated with 10 5 IU/ml IFN-α for 18 h before infection with VSV (MOI = 0.01). Viral load was determined at 48 h. All the results shown are representative of at least three independent experiments. All error bars indicate SEM.

Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

Techniques: Western Blot, Control, Two Tailed Test, Comparison, Quantitative RT-PCR, Infection

Journal: The Journal of Experimental Medicine

Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

doi: 10.1084/jem.20140280

Figure Lengend Snippet: Divergent responses to IL-6 in the TYK2-deficient patients. (A and B) Western blot of EBV–B cells from a healthy control (C1 and C2), TYK2-deficient patients (P1, P3, P5, P7, and P8), and an AD-HIES patient heterozygous for a STAT3 mutation (WT/S614G; STAT3*) after treatment with 50 ng/ml IL-6 for 20 min, probed with an Ab specific for phosphorylated STAT3 (pSTAT3). After analysis by densitometry, a p-value <0.05 (0.0332) for the two-tailed Student’s t test for the comparison of IL-6–pSTAT3 controls and P1 was found. However, a nonsignificant result was obtained for the comparison of IL-6–pSTAT3 controls and other than P1 TYK2-deficient patients. (C) EMSA with a GAS probe on EBV–B cells from a healthy control (C), TYK2-deficient patients (P1 and P2), a STAT1-deficient patient (STAT1*), and a heterozygous AD-HIES patient (WT/S614G for STAT3 ; STAT3*), without (−) and with (+) stimulation with 50 ng/ml IL-6 for 20 min. (B and C) White lines indicate intervening lanes were spliced out. (D) Western blot of primary fibroblasts from two healthy controls (C1 and C2), two TYK2-deficient patients (P1 and P2), and a patient with AD-HIES (STAT3*), with (+) or without (−) hIL-6 (30 ng/ml for 30 min) stimulation, probed with an Ab specific for phosphorylated STAT3 (pSTAT3). (A, B, and D) An anti-tubulin Ab was used as a loading control. (E) Western blot of proteins extracted from EBV–B cells from the TYK2-deficient patient P1 stably transduced with mock (P1 Mock ) or WT TYK2 (P1 TYK2 ) retroviral particles. Cells were (+) or were not (−) stimulated with 50 ng/ml IL-6 and/or 3.2 × 10 4 IU/ml IFN-β for 15 min, as indicated. Abs against TYK2, phosphorylated STAT3 (pSTAT3), STAT3, and tubulin were used. (F) Western blot of proteins extracted from EBV–B cells from the TYK2-deficient patient P1 stably transduced with retroviral particles encoding the scramble shRNA (Scr) or shRNA specifically targeting TYK2 (shRNA1 or 2). Cells were (+) or were not (−) stimulated with 50 ng/ml IL-6 for 20 min. Abs against TYK2, phosphorylated STAT3 (pSTAT3), STAT3, and tubulin were used. All the results shown represent at least two independent experiments. (G) Western blot of EBV–B cells from a control (C) and from P1 and P2, assessing the expression of components of the IL-6 pathway: JAK1, JAK2, gp130, IL-6Ra, and TYK2. (H) Naive and memory (defined as CD45RA − ) CD4 + T cells from WT controls ( n = 5) and TYK2-deficient patients ( n = 3; P1, P2, and P4) were purified (>98% purity) by FACS and cultured with TAE beads (anti-CD2/CD3/CD28) for 5 d, and the culture supernatants were then assessed for secretion of the cytokine indicated by cytometric bead assays or ELISA. Error bars indicate SEM.

Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

Techniques: Western Blot, Control, Mutagenesis, Two Tailed Test, Comparison, Stable Transfection, Transduction, Retroviral, shRNA, Expressing, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

doi: 10.1084/jem.20140280

Figure Lengend Snippet: TYK2 deficiency impairs the response to IL-23 but not to IL-27 nor IFN-γ. (A) Western blot analysis of STAT1 (pSTAT1, top) and STAT3 (pSTAT3, bottom) phosphorylation in EBV–B cells from healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an AD-HIES patient with a heterozygous STAT3 mutation (WT/T708N; STAT3*), after stimulation with 100 ng/ml IL-27 for 20 min. STAT1, STAT3, and α-tubulin levels were also assessed. The results shown are representative of at least two independent experiments. (B) Microarray analysis of HVS–T cell lines from three healthy controls, P2, and an IL-12Rβ1–deficient patient. Cells were stimulated for 12 h with 100 ng/ml IL-23. The difference between nonstimulated and stimulated cultures is shown as a fold change. (C) Western blot depicting phospho-STAT3 (pSTAT3) in EBV–B cells from a healthy control (C, C1, and C2), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), an AD-HIES patient carrying a heterozygous STAT3 mutation (WT/T708N; STAT3*), an IL-12Rβ1–deficient patient (IL-12Rβ1*), and a STAT1-deficient patient (STAT1*), without (−) and with (+) stimulation for 30 min with 100 ng/ml IL-23. α-Tubulin was used as a protein loading control. The results shown are representative of at least three independent experiments. After analysis by densitometry, a p-value <0.05 (0.0295) for the two-tailed Student’s t test for the comparison of IL-23–stimulated controls and TYK2-deficient patients (TYK2) was found. White lines indicate that intervening lanes have been spliced out. (D) Western blot depicting phospho-STAT3 (pSTAT3) in mock-transduced (left) and TYK2-transduced (right) EBV–B cells from a healthy control and P1, P2, P3, and P5, without (−) and with (+) stimulation for 30 min with 100 ng/ml IL-23. (E) Flow cytometry analysis of blood from TYK2-deficient patients P2 and P4, healthy controls (local controls, LC; and travel controls, TC), STAT3-deficient patients (STAT3*), and IL-12p40– and IL-12Rβ1–deficient patients (IL-12p40* and IL-12Rβ1*), showing the percentage of CD3 + T cells producing IL-17 ex vivo. A p-value <0.05 or <0.001 in two-tailed Student’s t tests is indicated by * or ***, respectively. Mean values for each set of conditions are indicated by solid lines. (F) Responses to IFN-γ (10 5 IU/ml for 20 min) and IL-27 (100 ng/ml for 20 min) were evaluated by EMSA in EBV–B cells from the TYK2-deficient patients (P1, P2, and P3), two healthy controls (C1 and C2), and a STAT1-deficient patient (STAT1*), with a GAS probe. (G) Flow cytometry analysis to assess the phosphorylation of STAT3 (pSTAT3) in EBV–B cells from two healthy controls (C1 and C2), three TYK2-deficient patients (P1, P2, and P3), and an AD-HIES patient (STAT3*), left untreated (−) or treated with 100 ng/ml IL-21 for 15 min. Intracellular staining was performed with APC-conjugated anti–human pSTAT3 Ab. (H and I) Cytokine production by in vitro–differentiated naive CD4 + T cells from control donors and TYK2-deficient patients (P1, P2, and P4). Naive (CD45RA + CCR7 + ) CD4 + T cells were purified from the PBMCs of WT controls ( n = 6) or TYK2-deficient patients P1, P2, and P4 and cultured for 5 d. Cells were cultured with TAE beads (anti-CD2/CD3/CD28) alone (Th0) or together with polarizing stimuli (TGFβ, IL-1β, IL-6, IL-21, IL-23, anti–IL-4, and anti–IFN-γ) to generate Th17 type cells. After 5 d, culture supernatants were assessed for the secretion of IL-17A or IL-17F with cytometric bead assays. (J) Naive and memory (defined as CD45RA − ) CD4 + T cells from WT controls ( n = 5) and TYK2-deficient patients ( n = 3; P1, P2, and P4) were purified (>98% purity) by FACS and cultured with TAE beads (anti-CD2/CD3/CD28) for 5 d, and the IL-17A + cells were then assessed by flow cytometry. All error bars indicate SEM.

Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

Techniques: Western Blot, Phospho-proteomics, Mutagenesis, Microarray, Control, Two Tailed Test, Comparison, Flow Cytometry, Ex Vivo, Staining, In Vitro, Purification, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

doi: 10.1084/jem.20140280

Figure Lengend Snippet: TYK2-deficient cells display an impaired response to IL-10 family cytokines. (A) The induction of IFIT1 transcription was analyzed by RT-qPCR after the treatment for 2 h with 20 ng/ml IL-29 or IL-28B of EBV–B cells from two healthy controls (C1 and C2), two TYK2-deficient patients (P1 and P2), a patient with complete STAT1 deficiency (STAT1*), and an IL-10R2–deficient patient (IL-10R2*). Results are normalized with respect to GUS levels. Error bars indicate SEM. (B) EMSA was performed with a GAS probe and EBV–B cells from a healthy control (C), TYK2-deficient patients (P1 and P2), and an IL-10R2–deficient patient (IL-10R2*), with (+) and without (−) stimulation with 50 ng/ml IL-10 for 20 min. (C) Western blot of EBV–B cells from a healthy control (C), TYK2-deficient patients (P1, P2, P3, P5, P7, and P8), and an IL-10R2–deficient patient (IL-10R2*) after treatment with 50 ng/ml IL-10 for 15 min, probed with an Ab specific for phosphorylated STAT3 (pSTAT3). An Ab against tubulin was used as a loading control. After analysis by densitometry, a nonsignificant p-value for the two-tailed Student’s t test for the comparison of IL-10–pSTAT3 controls and TYK2-deficient patients (TYK2) was found. (B and C) White lines indicate that intervening lanes were spliced out. (D) Western blot showing the detection of phospho-STAT3 (pSTAT3) in mock-transduced (left) or TYK2-transduced (right) EBV–B cells from a healthy control and P1, P2, P3, and P5, without (−) and with (+) stimulation for 30 min with 50 ng/ml IL-10. (E) SOCS3 induction was analyzed by RT-qPCR after 6 h of treatment with 50 ng/ml IL-10, in EBV–B cells from two healthy controls (C1 and C2), TYK2-deficient patients (P1 and P2), and an IL-10R2–deficient (IL-10R2*) patient. Results are normalized with respect to GUS . The results shown in A–E are representative of at least two independent experiments. (F) Impaired response to IL-10 in the macrophages of P2. The inhibition of TNF production in response to LPS and IL-10 was assessed by ELISA in macrophages from a travel control (C), P2’s relatives (mother, father, and sister), P2, and a patient with AD-HIES (STAT3*). (G and H) Western blots assessing the phosphorylation of STAT3 (pSTAT3) in healthy controls (C1 and C2), a TYK2-deficient patient (P2), an AD-HIES patient (STAT3*), and a patient with complete STAT1 deficiency (STAT1*), with (+) or without (−) LIF (100 ng/ml for 15 min). (G) In SV40-fibroblasts. (H) In primary fibroblasts. (I and J) Total CD4 and CD8 T cells (I) and naive (CCR7 + CD45RA + ), central memory (CCR7 + CD45RA − ; cmem), effector memory (CCR7 − CD45RA − ; emem), and revertant effector memory (CCR7 − CD45RA + ) T cells in CD4 + (J, left) and CD8 + T cells (J, right) from healthy controls (C) and the TYK2-deficient patients P1, P2 (tested three times), P4 (tested twice), and P5 (TYK2). Mean values for each set of conditions are indicated by solid lines.

Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

Techniques: Quantitative RT-PCR, Control, Western Blot, Two Tailed Test, Comparison, Inhibition, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: Epithelial EP4 plays an essential role in maintaining homeostasis in colon

doi: 10.1038/s41598-019-51639-2

Figure Lengend Snippet: Colon homeostasis is impaired in epithelial EP4-deficient mouse. ( A ) Schema of recombination in Villin-Cre; EP4 flox/flox mice. ( B ) Representative microscopic view of isolated crypts. Scale bars = 100 μm. ( C ) qRT-PCR analysis of EP4 mRNA levels in Villin-Cre (n = 5) and EP4 cKO (n = 5) colon crypts from the mice at 8 weeks of age. ( D ) Hematoxylin and eosin ( H , E ) staining of colon for Villin-Cre and EP4 cKO mice. Scale bars = 50 μm. ( E ) Crypt length in Villin-Cre and EP4 cKO mouse colons (n = 4). ( F ) Cell size of colonic epithelial cells located in the top of colon crypts in Villin-Cre and EP4 cKO mice (n = 3). ( G , H ) Alcian Blue staining and quantification in the colons from Villin-Cre and EP4 cKO mice at 8 weeks of age (n = 3). Scale bars = 50 μm. ( I ) Muc2 staining of colons in Villin-Cre and EP4 cKO mice at 8 weeks of age. Scale bars = 50 μm. ( J ) qRT-PCR analysis of Muc2 mRNA levels in Villin-Cre and EP4 cKO mice (n = 5). ( K ) Chromogranin A staining of colon in Villin-Cre and EP4 cKO mice at 8 weeks of age. Scale bars = 50 μm. ( L ) Quantification of ( K ) (n = 3) and mRNA expression levels of Chromogranin A in Villin-Cre and EP4 cKO mice (n = 4) analyzed by qRT-PCR on colon crypts. ( M ) Dclk1 staining of colon in Villin-Cre and EP4 cKO mice at 8 weeks of age. Scale bars = 50 μm. ( N ) Quantification of ( M ) (n = 3) and mRNA expression levels of Dclk1 in Villin-Cre and EP4 cKO colon crypts (n = 4) analyzed by qRT-PCR. Results are shown as mean ± SEM. *** P < 0.005.

Article Snippet: Primary antibodies used in this study were obtained from the indicated suppliers: rabbit anti-Dclk1 (ab31704, Abcam), rabbit anti-Chromogranin A (ab15160, Abcam), rat anti-Ki67 (652402, BioLegend), rabbit anti-Muc2 (SC15334, Santa Cruz), rabbit anti-cleaved Caspase 3 (9664S, Cell Signaling Technology), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rabbit anti-F4/80 (ab6640, Abcam), rat anti-CD4 (14-0041-85, eBioscience), rat anti-Gr1 (14-5931-85, eBioscience), mouse anti-Ctnnb1 (610153 BD Biosciences), rat anti-CD8 (550281, eBioscience), and rabbit anti-EP4 (ab133170, Abcam).

Techniques: Isolation, Quantitative RT-PCR, Staining, Expressing

Journal: Scientific Reports

Article Title: Epithelial EP4 plays an essential role in maintaining homeostasis in colon

doi: 10.1038/s41598-019-51639-2

Figure Lengend Snippet: Increased apoptosis in EP4 cKO colons. ( A , B ) Staining ( A ) and quantification ( B ) of Cleaved Caspase 3 (top), TUNEL (middle), and Single strand DNA staining (bottom) in Villin-Cre and EP4 cKO colons (n = 3). Scale bars = 50 μm. ( C ) mRNA expression levels of apoptotic markers in Villin-Cre and EP4 cKO colon crypts (n = 4) analyzed by qRT-PCR. ( D ) mRNA expression levels of an anti-apoptotic factor Bcl-2 in Villin-Cre and EP4 colon crypts (n = 4) analyzed by qRT-PCR. ( E ) Representative electron microscopic views of surface epithelial cells in Villin-Cre and EP4 cKO colons. Irregular apical cell surface (arrowhead), abnormal cell polarity, fewer mitochondria (arrow), and denatured mitochondria (circles) in EP4 cKO colons. Scale bars = 10 μm (top), 500 nm (bottom). Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Primary antibodies used in this study were obtained from the indicated suppliers: rabbit anti-Dclk1 (ab31704, Abcam), rabbit anti-Chromogranin A (ab15160, Abcam), rat anti-Ki67 (652402, BioLegend), rabbit anti-Muc2 (SC15334, Santa Cruz), rabbit anti-cleaved Caspase 3 (9664S, Cell Signaling Technology), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rabbit anti-F4/80 (ab6640, Abcam), rat anti-CD4 (14-0041-85, eBioscience), rat anti-Gr1 (14-5931-85, eBioscience), mouse anti-Ctnnb1 (610153 BD Biosciences), rat anti-CD8 (550281, eBioscience), and rabbit anti-EP4 (ab133170, Abcam).

Techniques: Staining, TUNEL Assay, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Epithelial EP4 plays an essential role in maintaining homeostasis in colon

doi: 10.1038/s41598-019-51639-2

Figure Lengend Snippet: Increased apoptosis in 3D-spheroid cultures of EP4 cKO colons. ( A ) Representative images of colon spheroids treated with or without EP4 antagonist for 48 h. 3D-culture images (left), ( H , E ) (middle), and Alcian blue staining (right). Scale bars = 200 μm (left, middle), 50 μm (right). ( B ) Diameter of spheroids treated with or without EP4 antagonist for 48 h (n = 29). ( C ) Quantification of Alcian blue-positive cells in ( A ) (n = 3). ( D ) Representative staining of Muc2/E-cadherin (left), Cleaved Caspase3 (middle), and Ki67 (right) on spheroids treated with or without EP4 antagonist for 48 h. Scale bars = 50 μm. ( E ) Quantification of Muc2, Cleaved Caspase 3, and Ki67 staining in ( D ) (n = 3). ( F ) Time-course images of spheroids generated from Villin-Cre and EP4 cKO colon crypts. Scale bars = 200 μm. ( G ) Quantification of day 2 spheroids in ( F ) (n = 50). Results are shown as mean ± SEM. * P < 0.05, *** P < 0.005, **** P < 0.001.

Article Snippet: Primary antibodies used in this study were obtained from the indicated suppliers: rabbit anti-Dclk1 (ab31704, Abcam), rabbit anti-Chromogranin A (ab15160, Abcam), rat anti-Ki67 (652402, BioLegend), rabbit anti-Muc2 (SC15334, Santa Cruz), rabbit anti-cleaved Caspase 3 (9664S, Cell Signaling Technology), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rabbit anti-F4/80 (ab6640, Abcam), rat anti-CD4 (14-0041-85, eBioscience), rat anti-Gr1 (14-5931-85, eBioscience), mouse anti-Ctnnb1 (610153 BD Biosciences), rat anti-CD8 (550281, eBioscience), and rabbit anti-EP4 (ab133170, Abcam).

Techniques: Staining, Generated

Journal: Scientific Reports

Article Title: Epithelial EP4 plays an essential role in maintaining homeostasis in colon

doi: 10.1038/s41598-019-51639-2

Figure Lengend Snippet: Gene expression profiling revealed the potential inflammatory status of EP4 cKO colons. ( A ) GSEA of transcriptomic data from microarray on EP4 cKO versus Villin-Cre colons. ( B ) KEGG pathways positively enriched in EP4 cKO mouse colons analyzed with DAVID. ( C ) Biological processes (GO) positively enriched in EP4 cKO mouse colons analyzed with DAVID. ( D ) Biological processes (GO) negatively enriched in EP4 cKO mouse colons analyzed with DAVID. ( E ) GSEA plots of enrichment of the indicated gene signatures in EP4 cKO versus Villin-Cre colons using the “Hallmarks” compilation from Molecular Signature Database (MSigDB, Broad Institute). NES, normalized enrichment score; FDR, false discovery rate. ( F ) EP4 mRNA levels in the indicated IBD datasets. Box and whiskers graphs indicate the median and the 25th and 75th percentiles, with minimum and maximum values at the extremes of the whiskers. * P < 0.05, ** P < 0.01.

Article Snippet: Primary antibodies used in this study were obtained from the indicated suppliers: rabbit anti-Dclk1 (ab31704, Abcam), rabbit anti-Chromogranin A (ab15160, Abcam), rat anti-Ki67 (652402, BioLegend), rabbit anti-Muc2 (SC15334, Santa Cruz), rabbit anti-cleaved Caspase 3 (9664S, Cell Signaling Technology), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rabbit anti-F4/80 (ab6640, Abcam), rat anti-CD4 (14-0041-85, eBioscience), rat anti-Gr1 (14-5931-85, eBioscience), mouse anti-Ctnnb1 (610153 BD Biosciences), rat anti-CD8 (550281, eBioscience), and rabbit anti-EP4 (ab133170, Abcam).

Techniques: Expressing, Microarray

Journal: Scientific Reports

Article Title: Epithelial EP4 plays an essential role in maintaining homeostasis in colon

doi: 10.1038/s41598-019-51639-2

Figure Lengend Snippet: Immune cell infiltration in EP4 cKO colons. ( A , B ) Staining for F4/80, CD4, CD8, and Gr-1 ( A ) in colons from Villin-Cre and EP4 cKO mice, and quantification ( B ; n = 3). Scale bars = 50 μm. ( C ) mRNA expression levels of indicated genes in Villin-Cre and EP4 cKO colons analyzed by qRT-PCR (n = 4). ( D ) mRNA expression levels of inflammatory cytokines in Villin-Cre and EP4 cKO colons analyzed by qRT-PCR (n = 4). ( E ) mRNA expression levels of chemokines in Villin-Cre and EP4 cKO colons analyzed by qRT-PCR (n = 4). Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Primary antibodies used in this study were obtained from the indicated suppliers: rabbit anti-Dclk1 (ab31704, Abcam), rabbit anti-Chromogranin A (ab15160, Abcam), rat anti-Ki67 (652402, BioLegend), rabbit anti-Muc2 (SC15334, Santa Cruz), rabbit anti-cleaved Caspase 3 (9664S, Cell Signaling Technology), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rabbit anti-F4/80 (ab6640, Abcam), rat anti-CD4 (14-0041-85, eBioscience), rat anti-Gr1 (14-5931-85, eBioscience), mouse anti-Ctnnb1 (610153 BD Biosciences), rat anti-CD8 (550281, eBioscience), and rabbit anti-EP4 (ab133170, Abcam).

Techniques: Staining, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Epithelial EP4 plays an essential role in maintaining homeostasis in colon

doi: 10.1038/s41598-019-51639-2

Figure Lengend Snippet: Inflammation was severely exacerbated in EP4 cKO mice in DSS-colitis model. ( A ) Strategy of experimental colitis with 2% DSS treatment. ( B ) Representative macroscopic views of the mouse anus at day 5 after starting DSS treatment. Yellow dashed circle denotes the active bleeding of EP4 cKO mice. ( C ) Hemooccult score at indicated time points in Villin-Cre (n = 8) and EP4 cKO mice (n = 7). ( D ) Colonoscopic images of distal or middle colons from Villin-Cre and EP4 cKO mice. Arrow denotes longitudinal erosion, arrowhead denotes ulcer, dotted line outlines inflammatory mucosa, and circle marks fibrotic region. ( E , F ) Representative macroscopic image ( E ) and length ( F ) of the colon from Villin-Cre and EP4 cKO mice at day 7 after starting DSS treatment. (n: Villin-Cre = 8 and EP4 cKO mice = 7). ( G ) H&E staining of colons from Villin-Cre and EP4 cKO mice at day 7 after starting DSS treatment. Dotted line outlines ulcerations. Scale bars = 50 μm. ( H ) Histlogical injury scores in Villin-Cre (n = 8) and EP4 cKO mice (n = 7) at day 7 after starting DSS treatment. ( I , J ) Staining ( I ) for F4/80, CD4, CD8, and Gr-1 of colons in Villin-Cre and EP4 cKO mice at day 7 after starting DSS treatment, and quantification ( J ). Scale bars = 50 μm. (n: Villin-Cre = 8 and EP4 cKO mice = 7). Results are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Primary antibodies used in this study were obtained from the indicated suppliers: rabbit anti-Dclk1 (ab31704, Abcam), rabbit anti-Chromogranin A (ab15160, Abcam), rat anti-Ki67 (652402, BioLegend), rabbit anti-Muc2 (SC15334, Santa Cruz), rabbit anti-cleaved Caspase 3 (9664S, Cell Signaling Technology), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rabbit anti-F4/80 (ab6640, Abcam), rat anti-CD4 (14-0041-85, eBioscience), rat anti-Gr1 (14-5931-85, eBioscience), mouse anti-Ctnnb1 (610153 BD Biosciences), rat anti-CD8 (550281, eBioscience), and rabbit anti-EP4 (ab133170, Abcam).

Techniques: Staining

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Fhl1 and Sdpr expression in normal epithelium and tumors from human breast, kidney, and prostate. Immunohistochemical staining was performed on normal and tumor human tissue. Fhl1 and Sdpr staining was evident in normal epithelial cells of the breast, kidney, and prostate. In contrast, both Fhl1 and Sdpr expression were suppressed in infiltrating mammary duct carcinoma, renal cell carcinoma, and prostate adenocarcinoma. (bar = 60 microns).

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Fhl1 and Sdpr expression in human breast, kidney, and prostate tumors

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Expressing

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Selection of genes affected by Fhl1. Microarray analysis was performed to identify probe sets representing genes with mean fold change differences of 2 or more with P < 0.05 by t‐test. (a) Fhl1 transfected Src transformed cells were compared with control transfectants. Of approximately 45 000 probe sets examined, genes represented by 459 were induced by Fhl1, whereas genes represented by 189 were suppressed. (b) Non‐transformed cells were compared with Src transformed cells to identify genes affected by Fhl1 that were correspondingly regulated by Src. Genes represented by 53 probe sets were induced by Fhl1 and suppressed by Src, whereas 48 were suppressed by Fhl1 and induced by Src. (c) Src‐transformed homozygous null Cas knockout cells transfected with Cas were compared with control transfectants to identify genes correspondingly regulated by Fhl1, Scr, and Cas. Genes represented by four probe sets were induced by Fhl1 and suppressed by Src and Cas, whereas genes represented by two probe sets were suppressed by Fhl1 and induced by Src and Cas.

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Selection, Microarray, Transfection, Transformation Assay, Control, Knock-Out

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Effects of Src and mitogen‐activated protein kinase (MAPK) activity on Fhl1 and Sdpr expression. (a) An equal amount of total protein (10 µg/lane) from non‐transformed and Src‐transformed NIH 3T3 cells expressing a Central Dogma (CD)‐tagged Sdpr–GFP fusion protein was analyzed by Western blotting with antiserum specific for green fluorescence protein (GFP), Sdpr, mitogen‐activated protein kinase kinase (MEK), MAPK, active MAPK (p‐MAPK), active Src (p‐Src), and β‐actin as indicated. (b) An equal amount of protein (100 µg) was immunoprecipitated from non‐transformed and Src‐transformed NIH 3T3 cells expressing a CD‐tagged Sdpr–GFP fusion protein, and then examined by Western blot analysis with GFP or Sdpr antiserum. (c) An equal amount of total protein (10 µg/lane) from non‐transformed and Src‐transformed embryonic mouse brain cells was analyzed by Western blotting with antiserum specific for Sdpr, Fhl1, MAPK, active MAPK (p‐MAPK), active Src (p‐Src), and β‐actin as indicated. (d) An equal amount of total protein (10 µg/lane) from non‐transformed and Src transformed embryonic mouse fibroblasts with or without Cas was analyzed by Western blotting with antiserum specific for Fhl1, MAPK, active MAPK (p‐MAPK), Cas, active Src (p‐Src), and β‐actin as indicated. Src‐transformed cells were also treated with the MEK blocker PD98059 to determine if Src requires MAPK to suppress Fhl1 and Sdpr expression. In all cases, more Fhl1 and Sdpr protein was present in non‐transformed cells than Src‐transformed cells independent of MAPK activity.

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Activity Assay, Expressing, Transformation Assay, Western Blot, Fluorescence, Immunoprecipitation

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Correlation of Fhl1 and Sdpr expression with Gleason stage of prostate tumors

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Expressing

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Fhl1 gene methylation in Src transformed cells. The transcriptional start site of Fhl1 (+1) lies in a CpG island of several hundred residues. PCR products from bisulfate‐treated DNA from non‐transformed and Src‐transformed cells were subcloned and sequenced. Data are shown as the percentage of each CpG sequence that was methylated (n = 5). More methylation of most of these sites was found in Src‐transformed cells than in non‐transformed cells.

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: Methylation, Transformation Assay, Sequencing

Journal: Cancer Science

Article Title: Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate

doi: 10.1111/j.1349-7006.2008.00816.x

Figure Lengend Snippet: Genes regulated by Fhl1

Article Snippet: Affinity‐purified (Pierce MicroLink Peptide Coupling Kit, Cat No: 20485) rabbit antiserum targeted against Fhl1 (Proteintech Group, S1009) or Sdpr (Proteintech Group, 90124) was applied to the sections and incubated at 4°C overnight in IHC blocking buffer.

Techniques: