Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop regulates the proliferation of glioma stem cells via the IL6-mediated JAK2/STAT3 signaling pathway

doi: 10.1186/s13046-021-01931-9

Figure Lengend Snippet: SRSF1 regulated the proliferation of GSCs by binding to circATP5B and upregulating circATP5B expression. a The relative expression of circATP5B after SRSF1 knockdown or overexpression was detected by qRT-PCR. (GSC406: p < 0.01; GSC201: p < 0.05; One-Way ANOVA). b , c The RIP assay was performed after SRSF1 knockdown ( b ) or overexpression ( c ), followed by qRT-PCR to detect the enrichment of circATP5B in GSCs. (GSC406: p < 0.001; GSC201: p < 0.01; One-Way ANOVA). d , e The RNA pull-down assays showed the SRSF1 protein immunoprecipitation with circATP5B as detected by western blotting. f MTS assays showed that SRSF1 knockdown or overexpression affected the cell viability of GSCs and was reversed by circATP5B overexpression or knockdown, respectively. (GSC406: p < 0.001; GSC201: p < 0.001; One-Way ANOVA). g The relative expression correlation between HOXB5 and SRSF1 in 70 cases of glioma patients were detected by qRT-PCR. (Total: r = 0.6606, p < 0.0001; Grade II: r = 0.4726, p = 0.0354; Grade III: r = 0.5013, p = 0.0107; Grade IV: r = 0.5526, p = 0.0042; Pearson’s correlation analyses). h The EDU assays showed that SRSF1 knockdown or overexpression affected the proliferation of GSCs and was reversed by circATP5B overexpression or knockdown, respectively. Scale bar = 100 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). j The neurospheres formation assays showed that SRSF1 knockdown or overexpression affected the relative size of the neurospheres of GSCs and was reversed by circATP5B overexpression or knockdown, respectively. Scale bar = 20 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). i and k Limiting dilution assays showed that SRSF1 knockdown or overexpression affected the neurosphere-forming capacity of GSCs and was reversed by circATP5B overexpression or knockdown, respectively (GSC406: p < 0.01; GSC201: p < 0.05; ELDA analysis; circles represent corresponding points, triangles mean the point is outside of the log fraction number wells). l Schematic diagram of the putative HOXB5 binding site in the 3′-UTR of SRSF1. m and n The luciferase reporter assays showed that HOXB5 knockdown or overexpression affected the luciferase activities of SRSF1 in GSCs. (GSC406: p < 0.001; GSC201: p < 0.001; One-Way ANOVA). o The ChIP qRT-PCR showed that HOXB5 bound to the promoter of SRSF1. (GSC406: p < 0.01; GSC201: p < 0.001; One-Way ANOVA). p , q , r qRT-PCR ( p ) and western blotting ( q, r ) showed the SRSF1 expression was affected after HOXB5 knockdown or overexpression in GSCs. (GSC406: p < 0.01; GSC201: p < 0.001; One-Way ANOVA). EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data were expressed as the mean ± SD (three independent experiments). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Firstly, the tumor tissues were embedded in paraffin, sliced into 4 mm sections, and labeled with primary antibodies as below: HOXB5 (1:100; #ab254882, Abcam), SRSF1 (1:100; #12929-2-AP, Proteintech), IL6 (1:100; #ab233551, Abcam), Ki67 (1:100; #ab92742, Abcam), CD133 (1:100; #ab216323, Abcam) and nestin (1:100; #ab105389, Abcam).

Techniques: Binding Assay, Expressing, Over Expression, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Luciferase, Plasmid Preparation, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop regulates the proliferation of glioma stem cells via the IL6-mediated JAK2/STAT3 signaling pathway

doi: 10.1186/s13046-021-01931-9

Figure Lengend Snippet: The SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop regulated glioma tumorigenesis in vivo. a Representative images showed the size of intracranial tumors in the coronal location of eight groups (negative control, circATP5B knockdown, miR-185-5p mimic, HOXB5 overexpression, SRSF1 overexpression, circATP5B knockdown combined with HOXB5 overexpression, miR-185-5p mimic combined with HOXB5 overexpression, SRSF1 overexpression combined with circATP5B knockdown in GSC406). Scale bar = 10 mm. b The measured tumor volumes among eight GSC406 groups are indicated. (* p < 0.05 vs. the negative control group; ## p < 0.05 vs. the circATP5B knockdown group, $$ p < 0.05 vs. the miR-185-5p group, && p < 0.05 vs. the SRSF1 overexpression group; Student’s t-test). c Kaplan-Meier survival curves showed that HOXB5 overexpression, SRSF1 overexpression, circATP5B knockdown combined with HOXB5 overexpression, miR-185-5p mimic combined with HOXB5 overexpression in GSC406 shortened the survival times of nude mice. At the same time, it prolonged the survival times after miR-185-5p mimic was transfected, circATP5B knockdown, and SRSF1 overexpression combined with circATP5B knockdown in GSC406. For each group, n = 5. d Representative immunohistochemical staining showing the changes in HOXB5, SRSF1, IL6, Ki67, CD133 and nestin in the negative control, circATP5B knockdown, miR-185-5p mimic, HOXB5 overexpression, SRSF1 overexpression, circATP5B knockdown combined with HOXB5 overexpression, miR-185-5p mimic combined with HOXB5 overexpression, SRSF1 overexpression combined with circATP5B knockdown group in orthotopic xenograft models. Scale bar = 50 μm. e The German scoring of HOXB5 protein expression in eight groups. (the circATP5B knockdown group vs. the negative control group: p < 0.001; the miR-185-5p mimic group vs. the negative control group: p < 0.001; the HOXB5 overexpression group vs. the negative control group: p < 0.01; the SRSF1 overexpression group vs. the negative control group: p < 0.01; the circATP5B knockdown combined with HOXB5 overexpression group vs. the negative control group: p < 0.01; the miR-185-5p mimic combined with HOXB5 overexpression group: p < 0.01; the SRSF1 overexpression combined with circATP5B knockdown group vs. the negative control group: p < 0.001; Student’s t-test). f Schematic diagram showing that the SRSF1/circATP5B/miR-185-5p/HOXB5 axis promoted glioma proliferation through IL6-mediated JAK2/STAT3 signaling pathway. All data were expressed as the mean ± SD (three independent experiments). EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data were expressed as the mean ± SD (three independent experiments). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Firstly, the tumor tissues were embedded in paraffin, sliced into 4 mm sections, and labeled with primary antibodies as below: HOXB5 (1:100; #ab254882, Abcam), SRSF1 (1:100; #12929-2-AP, Proteintech), IL6 (1:100; #ab233551, Abcam), Ki67 (1:100; #ab92742, Abcam), CD133 (1:100; #ab216323, Abcam) and nestin (1:100; #ab105389, Abcam).

Techniques: In Vivo, Negative Control, Over Expression, Transfection, Immunohistochemical staining, Staining, Expressing, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: An APEX2 proximity ligation method for mapping interactions with the nuclear lamina

doi: 10.1083/jcb.202002129

Figure Lengend Snippet: Related to : Additional analyses of our APEX2-lamin-B1–identified proteome. (A) A heatmap matrix representing the percentage overlap between the APEX2-lamin-B1 identified proteome and previously identified NL or nuclear membrane proteomes. We calculated the percentage between two datasets by dividing the overlapping number over the pooled, nonredundant datasets. HPA represents nuclear membrane localized proteins identified by the Human Protein Atlas Project. (B) Ranking of APEX2-lamin-B1 proteome normalized to tyrosine content. Red circles indicate known NL proteins, and blue circles represent PCBP proteins. (C) Density line plot of protein tyrosine content for nuclear protein (red line), cytosolic proteins (blue line), and our APEX2-lamin-B1–identified proteins (black line). Nuclear and cytosolic proteins were defined by the Human Protein Atlas Project. (D) Pie charts of our APEX2-lamin-B1 proteome showing the number of transmembrane (left) and secretory (right) proteins as defined by the Human Protein Atlas Project. (E) Western blot validation of SRSF1, SRSF2, and HNRNPA1 identified in our APEX2-lamin-B1 proteome. PrA is a protein-A dynabead control. (F) Immunostaining for PCBP2 and Nup153 in WT and lamin TKO mESCs. (G) Immunostaining of PCBP2 and lamin-A/C in WT, lamin-B1/B2-null (DKO), and DKO plus lamin-A/C siRNA SV-40–transformed MEFs. (H) Boxplot displaying the nuclear/cytosol ratio for the indicated MEF genotypes ( n ≥ 31). Notches represent the 95% confidence interval around the median, and blue dots represent the individual measurements. *, a two-tailed t test P value of <0.05. Int. dens, integrated density; MS, mass spectrometry.

Article Snippet: Detection reagents used were streptavidin-HRP (1:1,000; GE Healthcare; RPN1231V), rabbit anti-lamin-B1 (1:10,000; Abcam; ab16048), mouse anti-lamin-A/C (1:5,000; Active Motif; 39287), rabbit anti-emerin (1:5,000; Santa Cruz; sc-15378), mouse anti-SC-35 (1:2,500, Sigma-Aldrich; S4045), rabbit anti-HNRNPA1 (1:2,500; ProteinTech; 11176-1-AP), and rabbit anti-SRSF1 (1:2,500; ProteinTech; 12929-2-AP).

Techniques: Western Blot, Immunostaining, Transformation Assay, Two Tailed Test, Mass Spectrometry