Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 1. Biological activity of hLMP2 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#2 (T type) clone and flat revertants of the SKN-LMP2#122 (F type) clone (magnification 100). Changes in human uterine LMS cell line, SKN-transfectants, SKN-CEM9 (T type) clone, and SKN-LMP2wt (F type) clone xenograft volumes in mice (n = 8). Representative photographs of xenografts in mice (Left). Tumor growth of SKN-LMP2 was markedly reduced in comparison with that of the control transfectant SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9 (T type) clone and SKN-LMP2 (F type) clone (Right). (B) RT-PCR experiments revealed hLMP2, hLMP7, Calponin h1, SRF, cyclin B and b-actin mRNA expression in tumors. Precursor LMP2 or LMP7 (pre-LMP2, pre- LMP7) and mature LMP2 or LMP7 (LMP2, LMP7) are shown. (C) Western blotting revealed LMP2, LMP7, calponin h1, SRF, cyclin B, and b-actin in SKN-transfectant clones. (D) The luciferase reporter vectors containing the hCalponin h1 promoter with wild type SRF binding sites (Calponin-wt-Luc.), mutant SRF binding sites (Calponin-mut-Luc.), or empty luciferase reporter vector (Basic-Luc.) [23] were transiently co-transfected with pSV-b-galactosidase in SKN-transfectants, SKN-CEM9#2, SKN-LMP2#121, or SKN- LMP2#122 clones for the final 48 h, and then luciferase activities were measured. Values were normalized to those obtained with the co-transfected pSV-b-galactosidase expression vector. Each assay was performed at least three times and in triplicate. Luciferase reporter assays showed that LMP2 expression markedly induced calponin h1 promoter activation. Data are presented as the mean from three independent experiments (⁄S.D.). The experiments were performed four times with similar results. SKN transformantsa, CEM9 SKN-CEM9#2; LMP2, SKN-LMP2wt#121, SKN-LMP2wt#122. Detail is shown in SFig. 2, SFig. 3 and STable 3. RT-PCRb, total RNA samples were isolated from the individual xenografted-tumors, which were removed at 5 weeks after xenografting. W.B.c, W.B. are performed with the total cell lysates from SKN transformants.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, Comparison, Control, Transfection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Binding Assay, Mutagenesis, Plasmid Preparation, Activation Assay, Isolation

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 2. Biological activity of calponin h1 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#1Scr.shRNA (T type) clone, SKN-CEM9#2 calponin h1shRNA (T type) clone, SKN-CEM9#2 (T type) clone, SKN-LMP2#1Scr.shRNA (F type) clone, and SKN-LMP2#2Calponin h1shRNA (T type) clone of the SKN-LMP2 (F type) clone (magnification 60). The growth rates of the SKN-transfectant clones were measured as population doubling time (PDT). (B) Western blotting and RT-PCR experiments revealed calponin h1, precursor LMP2 (pre-LMP2), mature LMP2 (LMP2), and b-actin in SKN-transfectant clones. SKN transformantsa, CEM9#3 Scr.shRNA, CEM9#4 Calponin h1shRNA, LMP2#1 Scr.shRNA, LMP2#2 Calponin h1shRNA, Detail is shown in Table 1 and SFig. 5 and STable 3. (C) Changes in the human uterine LMS cell line, SKN-transfectant, SKN-CEM9#2 (T type) clone, SKN-LMP2wt#2/Calponin h1shRNA (T type) clone, and SKN-LMP2wt#1/ Scr.shRNA (F type) clone xenograft volumes in mice (n = 3). Representative photographs of xenografts in mice (Left). Tumor growth of the SKN-LMP2wt#2/Calponin h1shRNA (T type) clone is mildly increased in comparison with that of the SKN-LMP2wt#1/Scr.shRNA (F type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-transfectant clones (Right). RT-PCR experiments revealed hCalponin h1, hLMP2 and b-actin mRNA expression in tumors (Bottom). Experiments were performed three times with similar results. SKN-CEM9c, SKN- CEM9#2; LMP2wt+Calponin h1shRNAd, SKN-LMP2wt#2/ CalponinshRNA; LMP2wt/Scr.shRNAe, SKN-LMP2wt#1/Scr.shRNA. Details of SKN transfectants are shown in Table 1, SFig. 5 and STable 3. RT-PCRf, total RNA samples were isolated from the individual xenografted-tumors, which were removed from BALB/c nu/numice at 5 weeks after xenografting. Xenograftsg, BALB/c nu/nu mice were inoculated with SKN-CEM9#2, SKN-LMP2wt#2/CalponinshRNA or SKN-LMP2wt#1/Scr.shRNA.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, shRNA, Transfection, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Injection, Expressing, Isolation

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: Effect of NLRP3 siRNA on NLRP3 inflammasome activity and the downstream factors IL-18, IL-1β, GFAP and p-ERK1/2. (A) Transfection with NLRP3 siRNA significantly decreased the protein expression levels of caspase-1, ASC and NLRP3. (B) NLRP3 siRNA also significantly reduced IL-18 and IL-1β protein expression levels. (C) After microinjection of NLRP3 siRNA, the protein expression levels of p-ERK1/2 and GFAP significantly decreased. n=3 mice/group. * P<0.05 vs. saline + scramble; # P<0.05 vs. Coll IV + scramble. NLRP3, NLR family pyrin domain containing 3; siRNA, small interfering RNA; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Activity Assay, Transfection, Expressing, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: miR-223 targets the NLRP3 3'UTR and affects the protein expression levels of caspase-1, ASC, NLRP3, IL-18, IL-1β, p-ERK1/2 and GFAP. (A) Binding site of miR-223 within the NLRP3 3'-UTR. (B) miR-223 agomir significantly reduced the relative luciferase activity in PC12 cells transfected with the NLRP3 3'-UTR. (C) miR-223 agomir significantly increased miR-223 expression levels. (D) Administration of miR-223 agomir significantly reduced the protein expression levels of caspase-1, ASC and NLRP3. (E) miR-223 agomir also significantly reduced the expression of IL-18 and IL-1β. (F) After microinjection of miR-223 agomir, p-ERK1/2 and GFAP protein expression levels significantly decreased. n=3 mice/cohort. * P<0.05 vs. saline + agomir-scramble; # P<0.05 vs. Coll IV + agomir-scramble. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; UTR, untranslated region; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD; wt, wild-type; mut, mutant.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: Effect of miR-223 antagomir on the nociceptive threshold in naïve mice. Administration of miR-223 antagomir contributed to a significant increase in the paw withdrawal frequency in response to (A) 0.07 g and (B) 0.4 g von Frey filaments and a significant decrease in paw withdrawal latency to (C) thermal and (D) cold stimuli on day 3, 5 and 7 on the contralateral side. (E-G) Administration of miR-223 antagomir or antagomir-scramble did not contribute to behavioral changes on the ipsilateral side. n=8 mice/group. (H) miR-223 antagomir significantly reduced miR-223 expression levels. (I) Administration of miR-223 antagomir significantly elevated caspase-1, ASC and NLRP3 protein expression levels. (J) miR-223 antagomir also significantly elevated IL-18 and IL-1β protein expression levels. (K) Following microinjection of miR-223 antagomir, p-ERK1/2 and GFAP protein expression levels increased. n=3 mice/group. * P<0.05 vs. naïve group. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: CY 09 affects IL-18, IL-1β, p-ERK1/2 and GFAP protein expression levels. (A) CY 09 injection caused no alterations in miR-223 expression levels. CY 09 injection (B) significantly reduced IL-18 and IL-1β protein expression levels in the naïve + miR-223 antagomir cohort and (C) significantly lowered the protein expression levels of GFAP and p-ERK1/2. n=3 mice/cohort. * P<0.05 vs. naïve group; # P<0.05 vs. miR-223 antagomir + vehicle. GFAP, glial fibrillary acidic protein; p, phosphorylated; miR, microRNA.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing, Injection

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a – c Immunoblot analysis of FABP5 binding to ULCFA ( a ), SLCFA ( b ) or ULCFA with Grx1 treatment ( c ). Purified recombinant FABP5 protein was incubated with GSH plus H 2 O 2 or GSSG for 15 min, then mixed with Biotin-ULCFA or Biotin-SLCFA in the absence or presence of BMS309403 or linoleic acid or Grx1. FABP5 interacted with the Biotin-ULCFA or Biotin-SLCFA was captured using streptavidin magnetic beads. d Immunofluorescence staining of BMDMs after exposure to H 2 O 2 (200 μM) for 30 min (green, FABP5; blue, DAPI; scale bars, 10 μm). e Quantitative analysis of the ratio of nuclear/cytoplasmic fluorescence intensity in BMDMs as in ( d ), n = 10 in each group, P (ctr vs. ctr H2O2) = 0.0172, P (ctr H2O2 vs. Grx1 KO H2O2) <0.0001. f Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells. COS-7 cells were transfected with pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 followed by exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. g Immunoblots with antibodies against the indicated proteins in nuclear and cytoplasmic fractions from COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 (or vector) and pcDNA3.1-3xFlag-Grx1 (or vector) for 24 h, then exposed to H 2 O 2 (200 μM) for 1 h. h Immunoblots against the indicated proteins of cytoplasmic and nuclear extracts from BMDMs exposed to H 2 O 2 (200 μM) for 1 h. i mRNA expression of Il1b , Il6 , and Tnfα in BMDMs from mice stimulated with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr vs. Ctr+LPS) = 0.0013, P ( Il1b , Ctr+LPS vs. Grx1 KO+LPS) = 0.0091, *** P < 0.0001. j mRNA levels of Il1b , Il6 , and Tnfα in BMDMs pre-treated with an FABP5 inhibitor (SBFI26, 100 μM) for 2 h and stimulated with LPS (100 ng/mL) for 4 h, as determined by qPCR, n = 3 in each group, *** P < 0.0001. k mRNA levels of Il1b , Il6 , and Tnfα in BMDMs after treated with SBFI26 (100 μM) for 2 h and stimulation with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P ( Il1b , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Il16 , Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P ( Tnfα , Ctr+LPS vs. Ctr+LPS+inhibitor) = 0.0046. All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for (e, i-k). * P < 0.05, ** P < 0.01, *** P < 0.001, ns = no significance. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Western Blot, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads, Immunofluorescence, Staining, Fluorescence, Transfection, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

doi: 10.1038/s41467-021-27428-9

Figure Lengend Snippet: a Model structure of FABP5 and its 6 cysteines, generated by Pymol and based on the crystal structure of Homo sapiens FABP5 (PDB ID,4LKT). b Mass spectra of a peptide from FABP5 including glutathionylated cysteine 120 and cysteine127. c Immunofluorescence staining (with FABP5) and confocal microscopy imaging of COS-7 cells with overexpression of FABP5 WT, C67S, C87S, C120S, and C127S after exposure to H 2 O 2 (200 μM) for 1 h (green, FABP5; blue, DAPI; scale bars, 10 μm). d Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells transfected with FABP5 WT or C127S after exposure to H 2 O 2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. e Model structure of FABP5 interaction with an FA (Fatty Acid, Linoleic Acid), generated by Pymol and based on the crystal structure of FABP5 (PDB ID, 4LKT). f Docking structure of FABP5 in complex with S-glutathionylated Cys127 interacting with an FA (Fatty Acid, Linoleic Acid). g Docking structure of dimer FABP5 (PDB ID, 4AZM, Homo sapiens ) in complex with reduced glutathione (GSH) on Cys127. h Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or C127S and exposed to H 2 O 2 (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). i Immunoblot analysis of FABP5 fatty acid binding. Purified recombinant FABP5 WT or C127S protein (1 μg) was incubated with GSSG (1 mM) for 15 min, then mixed with Biotin-linoleic acid in the absence or presence of BMS309403 (5 mM) for 30 min. FABP5 that associated with the Biotin-linoleic acid was captured using streptavidin magnetic beads. Source data are provided as a Source Data file.

Article Snippet: Total cell lysates were used for Western blot using antibodies against FABP5 (Cell Signaling Technology, 39926, 1:1000), FABP5 (Proteintech, 12348-1-AP, 1:500), Grx1 (Abcam, ab45953, 1:250), β-actin (Huabio, M1210-2, 1:2000), PPARβ/δ (Santa Cruz, sc-74517, 1:500), Lamin A/C (Cell Signaling Technology, 4777, 1:2000) and Lamin B1 (Proteintech, 66059-1-Ig, 1:1000).

Techniques: Generated, Immunofluorescence, Staining, Confocal Microscopy, Imaging, Over Expression, Western Blot, Transfection, Co-Immunoprecipitation Assay, Expressing, Negative Control, Binding Assay, Purification, Recombinant, Incubation, Magnetic Beads