Journal: BMC Developmental Biology

Article Title: BMP-SMAD signalling output is highly regionalized in cardiovascular and lymphatic endothelial networks

doi: 10.1186/s12861-016-0133-x

Figure Lengend Snippet: The GFP reporter protein faithfully recapitulates the transcriptional activation of the BRE :: gfp transgene. In situ hybridisation for gfp mRNA ( red ) and direct GFP fluorescence ( green ) on cryosections through the cardinal vein ( a – d ) and the ventricular trabeculae ( e – g ) at E11.5. DAPI is used to stain nuclei. The boxed area in ( a ) is enlarged in panels ( b – d ). The corresponding low magnification view of the trabeculae in the ventricle is not provided because of insufficient intensity of the ISH signal. White arrowheads indicate ECs double positive for gfp mRNA and direct GFP fluorescence. The red and green arrows depict ECs only positive for gfp mRNA or GFP fluorescence respectively. Scale bars: 100 μm ( a ); 50 μm ( b – d ); 10 μm ( e – g )

Article Snippet: The GFP fluorescent in situ hybridisation (ISH) probe was custom designed with the probe designer tool from Stellaris (LGC biosearch technologies).

Techniques: Activation Assay, In Situ, Hybridization, Fluorescence, Staining

Journal: Frontiers in Microbiology

Article Title: AC5 protein encoded by squash leaf curl China virus is an RNA silencing suppressor and a virulence determinant

doi: 10.3389/fmicb.2022.980147

Figure Lengend Snippet: GFP silencing in GFP-transgenic N . benthamiana 16c plants. (A) Representative leaf patches or plants were co-infiltrated with A . tumefaciens cultures harboring the p35S-GFP, p35S-AC5, and expression vectors pGreenII 62-SK or the p19 vector. Images were photographed under UV light at 4 dpi. (B) Northern blot of GFP and AC5 mRNA accumulation, western blot of GFP protein accumulation and siRNA blot of GFP siRNA accumulation in Agrobacterium -infiltrated leaf patches as indicated in (A) . SYBR Safe DNA Gel staining of rRNA and Ponceau S staining of the subunit of Rubisco served as loading controls. U6 was the loading control in the siRNA blot.

Article Snippet: Membranes were hybridized with a DIG-labeled GFP siRNA probe (Sangon Biotech Co., Ltd.).

Techniques: Transgenic Assay, Expressing, Plasmid Preparation, Northern Blot, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: AC5 protein encoded by squash leaf curl China virus is an RNA silencing suppressor and a virulence determinant

doi: 10.3389/fmicb.2022.980147

Figure Lengend Snippet: Identification of the AC5 region with RNA silencing suppressor activity. (A) Diagrams of the AC5 mutants. (B) Leaf patches were co-infiltrated with A . tumefaciens cultures harboring the p35S-GFP and empty pGreenII 62-SK vector, p35S-GFP + p35S-AC5, p35S-GFP + p35S-AC5(1–132), p35S-GFP + p35S-AC5(135–187), p35S-GFP + p35S-AC5(188–519), p35S-GFP + P19, p35S-AC5 and its mutants were photographed under UV light at 4 dpi. (C) Northern blot of GFP accumulation, GFP protein accumulation and siRNA blot of GFP siRNA accumulation in the Agrobacterium -infiltrated leaf patches as indicated in (B) . SYBR Safe DNA Gel staining of rRNA and Ponceau S staining of the subunit of Rubisco served as loading controls. U6 was the loading control in the siRNA blot.

Article Snippet: Membranes were hybridized with a DIG-labeled GFP siRNA probe (Sangon Biotech Co., Ltd.).

Techniques: Activity Assay, Plasmid Preparation, Northern Blot, Staining

Journal: Frontiers in Microbiology

Article Title: AC5 protein encoded by squash leaf curl China virus is an RNA silencing suppressor and a virulence determinant

doi: 10.3389/fmicb.2022.980147

Figure Lengend Snippet: The SLCCNV AC5 could not inhibit GFP silencing of double-stranded dsRNA. (A) Representative 16c leaves were co-infiltrated with A . tumefaciens cultures harboring the p35S-GFP, double-stranded GFP (p35S-dsFP) and vector pGreenII 62-SK, p35S-AC5-expressing vectors or p19. Images were photographed under UV light at 4 dpi. (B) Northern blot of GFP and AC5 mRNA accumulation, western blot of GFP protein accumulation, and siRNA blot of GFP siRNA accumulation in Agrobacterium -infiltrated leaf patches. Ethidium bromide staining of rRNA and Ponceau S staining of the subunit of Rubisco served as loading controls. U6 was the loading control in the siRNA blot.

Article Snippet: Membranes were hybridized with a DIG-labeled GFP siRNA probe (Sangon Biotech Co., Ltd.).

Techniques: Plasmid Preparation, Expressing, Northern Blot, Western Blot, Staining

Journal: Communications Biology

Article Title: Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma

doi: 10.1038/s42003-024-06918-8

Figure Lengend Snippet: A MALAT1 levels were significantly increased in MM samples. Patients were designated healthy donors with normal BM plasma cells (NP, n = 22) or multiple myeloma (MM, n = 559). B FISH analysis of MALAT1 expression in BM tissues from healthy donors ( n = 4) and BM tissues from MM patients ( n = 36) (plotted as mean ± s.d. after normalization to the control group). Scar bar = 20 μm. C Intercellular trafficking of MALAT1 packaged into exosomes and taken up by RAW264.7 cells by isolated U266 exosomes labeled with PKH26 dye and MALAT1 labeled with GFP and cultured with RAW246.7 cells. The nuclei of RAW264.7 cells were stained with DAPI (blue) ( n = 3 independent experiments). Scar bar = 25 μm. D , E Relative MALAT1, and RANKL RNA expression in RAW264.7 cells were detected by qRT-PCR ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control group). The groups were as follows: Control: Cells treated with sRANKL alone. EXO: Cells treated with sRANKL and exosomes derived from untreated U266 cells. EXO-siNC: Cells treated with sRANKL and exosomes derived from U266 cells transfected with a negative control siRNA. EXO-siMALAT1: Cells treated with sRANKL and exosomes derived from U266 cells transfected with siRNA targeting MALAT1. EXO-Vector: Cells treated with sRANKL and exosomes derived from U266 cells transfected with an empty vector. EXO-MALAT1: Cells treated with sRANKL and exosomes derived from U266 cells transfected with a vector overexpressing MALAT1. F Western blot results of RANKL in RAW264.7 cells ( n = 3 independent experiments). G TRAP staining of RAW264.7 cells. TRAP-positive multinucleated cells containing more than three nuclei were counted and analyzed ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to the control group). Scar bar = 50 μm. P -value by Welch’s t -test ( A , B ), or one-way ANOVA followed by Tukey’s multiple comparison test ( D , E , G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: GFP-labeled MALAT1 probes and CY3-labeled MALAT1 probes were obtained from Beijing Dingguo Changsheng Biotech Co., Ltd., were synthesized for FISH analysis.

Techniques: Clinical Proteomics, Expressing, Control, Isolation, Labeling, Cell Culture, Staining, RNA Expression, Quantitative RT-PCR, Derivative Assay, Transfection, Negative Control, Plasmid Preparation, Western Blot, Comparison

Journal: Communications Biology

Article Title: Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma

doi: 10.1038/s42003-024-06918-8

Figure Lengend Snippet: A An RNA pulldown experiment was conducted to examine the interaction between NSUN2 and MALAT1. Antisense MALAT1 was used as a negative control ( n = 3 independent experiments). B The m5C MeRIP assay followed by qRT-PCR was used to assess the m5C level of MALAT1 in U266 cells transfected with control or NSUN2 siRNAs ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to input RNA levels and IgG group). C siRNA targeting NSUN2 was transfected into U266 cells, and MALAT1 was tested using qRT-PCR ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and siNC group). D RIP assays using NSUN2 antibody. IgG was used as a negative control ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to input RNA levels and IgG group). E U266 cells transfected with control or NSUN2 siRNAs were exposed to actinomycin D (5 μg/mL), and cellular RNA was isolated at the indicated times. qRT-PCR was performed to assess the half-life of MALAT1 ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to the 0-hour time point). F siRNA targeting YBX1 was transfected into NSUN2-overexpressing U266 cells, and the expression level ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control group). G The stability of MALAT1 in NSUN2-overexpressing U266 cells transfected with YBX1 siRNA was assessed ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to the 0-hour time point). H NSUN2 and YBX1 RNA levels were significantly increased in MM samples. Patients were designated healthy donors with normal BM plasma cells (NP, n = 22) or multiple myeloma (MM, n = 559). I Representative IHC images of healthy donors ( n = 3) and MM patients ( n = 36) BM locating NSUN2 and YBX1 (plotted as mean ± s.d. after normalization to the control group). Scar bar = 50 μm. Colorectal cancer tissue was used as a positive control. Negative controls were bone marrow tissues stained with the primary antibody omitted and replaced with PBS (Additional file 3). P -value by two-tailed Student’s t -test ( B – E , H ), Welch’s t -test ( I ), or one-way ANOVA followed by Tukey’s multiple comparison test ( F , G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ▲ P < 0.05 versus the NSUN2-OE group.

Article Snippet: GFP-labeled MALAT1 probes and CY3-labeled MALAT1 probes were obtained from Beijing Dingguo Changsheng Biotech Co., Ltd., were synthesized for FISH analysis.

Techniques: Negative Control, Quantitative RT-PCR, Transfection, Control, Isolation, Expressing, Clinical Proteomics, Positive Control, Staining, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma

doi: 10.1038/s42003-024-06918-8

Figure Lengend Snippet: To study the correlation between MALAT1, RANKL, YBX1, NSUN2, and bone lesion formation, we detected their expression in BM tissues of NDMM patients ( n = 36) by FISH or IHC. MALAT1 expression positively correlated with bone lesion formation ( A ), corrected serum calcium ( B ), and RANKL level ( C ). D MALAT1 expression positively correlated with YBX1 and NSUN2 levels. NSUN2 ( E ) and YBX1 ( F ) expression positively correlated with bone lesion formation, RANKL level, and corrected serum calcium ( G ). H The accuracy of MALAT1, NSUN2, and YBX1 in predicting the death outcome of MM patients with bone lesions. I Representative IHC (scar bar = 50 μm) and FISH (scar bar = 10 μm) images of high-risk and low-risk MM patients ( n = 36) BM locating NSUN2, YBX1, and MALAT1 (plotted as mean ± s.d. after normalization to low-risk group). For FISH, MALAT1 expression was assessed specifically in myeloma cells by co-staining with CD138. For IHC, colorectal cancer tissue was used as a positive control. Negative controls were bone marrow tissues stained with the primary antibody omitted and replaced with PBS (Additional File 3). J Cumulative survival function of overall survival analyses comparing MALAT1, NSUN2, and YBX1 high-risk and low-risk groups ( P < 0.01). P -value by Welch’s t -test ( I ). Pearson correlation analyses ( A – G ). Log-rank test ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: GFP-labeled MALAT1 probes and CY3-labeled MALAT1 probes were obtained from Beijing Dingguo Changsheng Biotech Co., Ltd., were synthesized for FISH analysis.

Techniques: Expressing, Staining, Positive Control

Journal: Communications Biology

Article Title: Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma

doi: 10.1038/s42003-024-06918-8

Figure Lengend Snippet: The univariate COX regression analysis for the potential prognostic factors of survival of MM patients with bone lesions

Article Snippet: GFP-labeled MALAT1 probes and CY3-labeled MALAT1 probes were obtained from Beijing Dingguo Changsheng Biotech Co., Ltd., were synthesized for FISH analysis.

Techniques:

Journal: Communications Biology

Article Title: Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma

doi: 10.1038/s42003-024-06918-8

Figure Lengend Snippet: A Relative expression levels of miR-17, miR-34a, miR-146a, miR-125a, and miR-101-1 were measured in RAW264.7 cells under various treatment conditions by qRT-PCR ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to U6 and control group). The groups were as follows: Control: Cells treated with sRANKL alone. EXO: Cells treated with sRANKL and exosomes derived from untreated U266 cells. EXO-siNC: Cells treated with sRANKL and exosomes derived from U266 cells transfected with a negative control siRNA. EXO-siMALAT1: Cells treated with sRANKL and exosomes derived from U266 cells transfected with siRNA targeting MALAT1. EXO-Vector: Cells treated with sRANKL and exosomes derived from U266 cells transfected with an empty vector. EXO-MALAT1: Cells treated with sRANKL and exosomes derived from U266 cells transfected with a vector overexpressing MALAT1. B The direct interaction of miR-17 with MALAT1 and RANKL was verified by dual-luciferase reporter assays ( n = 4 independent experiments; plotted as mean ± s.d. after normalization to the control group). C Western blot results of RANKL in RAW264.7 cells treated with sRANKL, sRANKL + U266 exosomes, sRANKL + U266-siNC exosomes + miR-NC inhibitors, sRANKL + U266-siMALAT1 exosomes + miR-17 inhibitors, sRANKL + U266-vector exosomes + miR-NC mimics or sRANKL + U266-MALAT1 exosomes + miR-17 mimics, and densitometric analysis of protein expression ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to β-actin and negative control group). D The mRNA expression of RANKL ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to GAPDH and control group). E Cells were fixed, subjected to TRAP staining, and observed under a light microscope. TRAP-positive multinucleated cells containing more than three nuclei were counted and analyzed ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to the control group). Scar bar = 50 μm. P -value by two-tailed Student’s t -test ( B ), or one-way ANOVA followed by Tukey’s multiple comparison test ( A , C – E ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: GFP-labeled MALAT1 probes and CY3-labeled MALAT1 probes were obtained from Beijing Dingguo Changsheng Biotech Co., Ltd., were synthesized for FISH analysis.

Techniques: Expressing, Quantitative RT-PCR, Control, Derivative Assay, Transfection, Negative Control, Plasmid Preparation, Luciferase, Western Blot, Staining, Light Microscopy, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma

doi: 10.1038/s42003-024-06918-8

Figure Lengend Snippet: A FISH analysis of MALAT1 expression in femur sections of mice from different groups ( n = 6 independent experiments; plotted as mean ± s.d. after normalization to the control group). Scar bar = 20 μm. B IHC analysis was used to detect the presence of p-p38, p-ERK, p-JNK, p-AKT, and RANKL in femur sections collected from the indicated groups of mice ( n = 3 independent experiments; plotted as mean ± s.d. after normalization to the control group). Scar bar = 20 μm. Representative images are shown for each group. P -value by one-way ANOVA followed by Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: GFP-labeled MALAT1 probes and CY3-labeled MALAT1 probes were obtained from Beijing Dingguo Changsheng Biotech Co., Ltd., were synthesized for FISH analysis.

Techniques: Expressing, Control, Comparison

Journal: Nature Plants

Article Title: Multiplexed single-cell 3D spatial gene expression analysis in plant tissue using PHYTOMap

doi: 10.1038/s41477-023-01439-4

Figure Lengend Snippet: The mRNA of GFP was targeted with HCR (left) or PHYTOMap (right) in ( a ) ELTP:FLS2-GFP and ( b ) LBD16::FLS2-GFP plants, which express GFP in endodermis and pericycle, respectively. Scale bars = 25 μm. c, PHYTOMap images that cover larger areas. (Left) ELTP:FLS2-GFP . (Right) LBD16::FLS2-GFP . Scale bar = 100 μm. Three independent roots were tested with similar results.

Article Snippet: After protein digestion and post fixation, the sample was pre-incubated in HCR probe hybridization buffer (Molecular Instruments, catalogue no. BPH02323) for 30 min at 37 °C, then incurvated in HCR probe hybridization buffer with a 1:500 volume of a GFP-targeting probe mixture (designed by Molecular Instruments) overnight at 37 °C.

Techniques: