Journal: Cell Death & Disease

Article Title: Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer

doi: 10.1038/s41419-021-03963-7

Figure Lengend Snippet: A The heatmap presents differentially expressed proteins between two pairs of exosomes (231-S-exo and 231-R-exo, 231-HM-S-exo and 231-HM-R-exo). B The Venn diagram of overlap between 231-R-exo/231-S-exo and 231-HM-R-exo/231-HM-S-exo. C , D TFPI, COL8A1, annexin A1, ANXA6 and HLA-B protein levels in MDA-231, MDA-231-R, MDA-231-HM, and MDA-231-HM-R cells and in 231-S-exo, 231-R-exo, 231-HM-S-exo, and 231-HM-R-exo. E The changes of ANXA6 in MDA-231 and MDA-231-HM cells treated with PBS, sensitive cell-derived exosomes (231-S-exo or 231-HM-S-exo), or resistant cell-derived exosomes (231-R-exo or 231-HM-R-exo). All the experiments were repeated three times, and the representative results are presented.

Article Snippet: The following antibodies were used: anti-ANXA6 (1:500, 12542-1-AP, Proteintech, USA), anti-ANXA1 (1:1000, 21990-1-AP, Proteintech, USA), anti-EGFR (1:1000, ab52894, Abcam, USA), anti-TFPI (1:2000, 66842-1-Ig, Proteintech, USA), anti-COL8A1 (1 µg/ml, ab100988, Abcam, USA), anti-HLA-B (1:1000, 17260-1-AP, Proteintech, USA), anti-Flag (ab205606, 1:1000, Abcam, USA) and anti-fibronectin (ab2413, 1:3000, Abcam, USA).

Techniques: Derivative Assay

Journal: Nucleic Acids Research

Article Title: The integrity of the U12 snRNA 3′ stem–loop is necessary for its overall stability

doi: 10.1093/nar/gkab048

Figure Lengend Snippet: The 84C>U mutation leads to destabilization of the U12 snRNA. ( A ) Northern blot analysis of spliceosomal snRNA levels in RNU12 +/+ and RNU12 84T/84T cells. Shown are three independent samples from RNU12 +/+ HEK293 cells (lanes 1–3) and samples from three different RNU12 84T/84T HEK293 single-cell clones (lanes 4–6). The same northern blot membrane was sequentially stripped and reprobed with different individual snRNA probes. For data visualization purposes, the signal intensity in the entire membrane (containing both RNU12 +/+ and RNU12 84T/84T samples) was adjusted so that the RNU12 +/+ lanes showed similar intensities between the different snRNAs. ( B ) Quantification of snRNA northern blot data from A . The quantification is based on measurements from three total RNA samples from RNU12 +/+ HEK293 cells, three samples from RNU12 84T/+ HEK293 single-cell clones and eight samples from RNU12 84T/84T HEK293 single-cell clones. Loading was normalized by dividing all band intensities by the sum of U1 and U2 intensities averaged across all samples and the average intensity of the WT samples was then set to 1 for each snRNA. Error bars represent standard deviation. Statistical significance levels (ns: P > 0.05, [∗] P ≤ 0.05, [∗∗] P ≤ 0.01, [∗∗∗] P ≤ 0.001) from one-way ANOVA followed by Tukey's test are indicated. ( C ) Levels of exogenously expressed WT and 84C>U-mutant U12 snRNA in HeLa, U2OS and HEK293 cell lines. Tag-U12 constructs or empty pUC19 vector were co-transfected into each cell line together with a plasmid for expressing the F30-2xdBroccoli aptamer used as a transfection control. ( D ) Actinomycin D treatment of RNU12 +/+ and RNU12 84T/84T HEK293 cells. Cells were treated with 5 μg/ml actinomycin D for 0–8 h and harvested at the indicated time points. Shown are northern blots probed with U12 and U6atac snRNA-specific probes. ( E ) Decay kinetics of the U12 and U6atac snRNAs in RNU12 +/+ and RNU12 84T/84T HEK293 cells upon actinomycin D treatment. The individual time series were normalized by setting the mean value of zero hour time point to 1.0. Statistical significance levels, determined using Student's t -test, refer to comparisons between U12 WT and U12 84C>U at the indicated time points.

Article Snippet: For tag-U12 experiments, each pUC19-RNU12-5′tag construct was cotransfected at a 3:1 ratio with pAVU6+27-F30-2xdBroccoli (Addgene #66842), a gift from Dr Samie Jaffrey. siRNA reverse transfections were carried out using Lipofectamine RNAiMAX (Thermo Fisher) and 30 nM final concentration of siRNA, followed by a 48 h incubation before RNA extraction with Trizol. siRNAs were purchased from Dharmacon or Sigma-Aldrich and their sequences are listed in .

Techniques: Mutagenesis, Northern Blot, Clone Assay, Standard Deviation, Construct, Plasmid Preparation, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: The integrity of the U12 snRNA 3′ stem–loop is necessary for its overall stability

doi: 10.1093/nar/gkab048

Figure Lengend Snippet: Identification and characterization of truncated U12 snRNAs in human cells. ( A ) Effects of mutations in the terminal base-pair of U12 snRNA stem-loop III on full-length U12 and U12 fragment levels. Top: Plasmid constructs for expressing tag-U12 with various combinations of nucleotides in the 84 and 150 positions were transfected into HeLa cells. Co-transfected F30-2xdBroccoli expression plasmid served as a transfection control. Total RNA from transfected HeLa cells was analyzed by northern blot with U12, U11, tag-U12 and Broccoli probes. Bottom: Quantification of full-length U12 (blue) and U12 fragment (orange) levels. Band intensities were normalized by the sum of all band intensities of each replicate, and the mean full-length U12 snRNA signal of WT tag-U12 (C-G) was then set to 1. Statistical significance was determined using one-way ANOVA followed by Dunnett's test. Significance levels above each bar refer to comparisons between WT tag-U12 and the indicated variant. Based on pair-wise comparisons (ANOVA with Tukey's test), differences in fragment levels between the group of variants showing low levels of tag-U12 fragments (C-G, G-C, G-G) and the group with high levels of fragments (U–G, U–A, C–C, U–C, C–A) were all statistically significant (*–***). In contrast, none of the pair-wise comparisons within the two groups showed statistically significant differences in fragment levels. ( B ) Location of truncation sites of U12 snRNA fragments determined by Sanger sequencing. See for details. ( C ) Detection of short U12 snRNA species in association with Sm proteins. Forty eight hours after control, MTR4 or RBM7 siRNA transfection, anti-Sm immunoprecipitations were carried out using total cell lysates from RNU12 +/+ and RNU12 84T/84T cells. RNA extracted from input and IP samples was analyzed by northern blotting with a probe specific to U12 (1–31). The U1 and 7SK snRNA were also analyzed as controls. The anti-Sm antibody immunoprecipitated the U1 snRNA, whereas the 7SK snRNA, which does not associate with Sm proteins, was only weakly detected. ( D ) U12 snRNA fragments carry a trimethylated 5′ cap. Anti-TMG immunoprecipitation was carried out using total RNA from RNU12 +/+ and RNU12 84T/84T cells transfected with control or MTR4 siRNA. Northern blotting was carried out with U12 (1–31) and U2 snRNA (35–66) probes. ( E ) Cytoplasmic–nuclear distribution of U12 snRNA fragments. RNU12 84T/84T were fractionated into total (T), cytoplasmic (C) and nuclear (N) fractions and RNA from the fractions analyzed by northern blot and RT-PCR as indicated. tRNA(Glu) served as a marker for the cytoplasm, while the nuclear-retained lncRNA MALAT1 and the long-3′UTR isoform of the RNPC3 mRNA were used as nuclear markers.

Article Snippet: For tag-U12 experiments, each pUC19-RNU12-5′tag construct was cotransfected at a 3:1 ratio with pAVU6+27-F30-2xdBroccoli (Addgene #66842), a gift from Dr Samie Jaffrey. siRNA reverse transfections were carried out using Lipofectamine RNAiMAX (Thermo Fisher) and 30 nM final concentration of siRNA, followed by a 48 h incubation before RNA extraction with Trizol. siRNAs were purchased from Dharmacon or Sigma-Aldrich and their sequences are listed in .

Techniques: Plasmid Preparation, Construct, Expressing, Transfection, Northern Blot, Variant Assay, Sequencing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Marker