Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: (A) Graph representing the distribution of FUS-P525L protein in nucleus and cytoplasm. The fluorescent intensities of the nucleus and cytoplasm were calculated from the experiment as performed in , and the ratio is plotted here. A student-paired t-test was used to calculate the significance value (n=6). (B) Incucyte images (real-time cell death analysis) representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. (C) Graph representing the number of propidium iodide (PI) positive cells (dead cells) from the experiment as performed in B. The time on the x-axis reflects the time-point after transfection in hours. The significance was calculated using 2-way ANOVA with multiple comparisons (n=4). Error bars in all graphs represent mean ±SEM, and the same color points in A depict the data from a single experimental set. *, **, ***, and **** denote p-value≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively.

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: Transfection

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: Incucyte images representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. The images are part of and .

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques:

Journal: Carcinogenesis

Article Title: Expression of estrogen receptors in a normal human breast epithelial cell type with luminal and stem cell characteristics and its neoplastically transformed cell lines.

doi: 10.1093/carcin/18.2.251

Figure Lengend Snippet: Fig. 7. The proteins from cell lines used in experiments presented in Fig. 6 also were used for Western blot analysis for the expression of PgR. The PgR expression in these cells before innoculation for tumor growth in nude Fig. 6. Western blot analysis for the expression of ER. The expression of mice (lanes 2–5) and after re-establishment of cell culture from tumors ER in weakly tumorigenic cell line, M13SV1R2 and highly tumorigenic cell (lanes 10–12) were compared with that in tumors formed by these cells lines, M13SV1R2-N1, -4, -8, before innoculation for tumor growth in nude (lanes 6–9). The cell lysate loaded are: lanes 2, 6, 10, M13SV1R2; lanes 3, mice (lanes 1–4) and after reestablishment of cell culture from tumors 7, 11, M13SV1R2-N1; lanes 4, 8, 12, M13SV1R2-N4; lanes 5, 9, 13, (lanes 9–11), were compared with that in tumors formed by these cells M13SV1R2-N8. (lanes 5–8). The cell lysates loaded are; lanes 1, 5, 9, M13SV1R2; lanes 2, 6, 10, M13SV1R2-N1; lanes 3, 7, 11, M13SV1R2-N4; lanes 4, 8, M13SV1R2-N8. The positions of wild type ER (WT) and variant ER (VT) stem cell characteristics examined while their Type II HBEC are indicated. counterparts with basal cell characteristics did not express any ER. Furthermore, all the SV40 transformed Type I and Type preparation). We have tested the expression of ER in these II HBEC lines examined (15 and 11 cell lines, respectively) tumorigenic cell lines before innoculation in nude mice, in were also found to express the ER. Although the human tumors formed by these cells in nude mice and in cell cultures mammary gland is known to contain a small population of reestablished from tumors. The results showed that cells, ER-positive cell (26), the previously reported normal HBEC before injection into nude mice, and cells, reestablished from in culture have not been shown to express significant level of tumor tissue, in vitro, only expressed the variant ER (~48 kd) ER. The expression of ER in our Type I HBEC or SV40 using an anti-ER antibody recognizing the C-terminal region transformed cells is unambiguous since it has been observed of the ER (Ab-1, Oncogene Science). Significantly, the tumor by three different methods (i.e. Western blot analysis, immuno- tissues expressed a high level of the wild type ER (~66 kd) fluorescence staining, and RT-PCR). and less amount of the variant ER (~48 kd) by Western blotting The ER expression, however, is not the wild type ER. It is using the Ab-1 (Oncogene Science) (Figure 6) or the human ER specific antibody (D75, kindly provided by Dr Geoffrey a variant ER with smaller molecular weight (~48 kd) than the Greene of the University of Chicago) (data not shown). Western wild type ER (~66 kd). This variant ER was detectable by blot analysis for the expression of progesterone receptor using Western blot analysis and immunofluorescence staining using an anti-PgR antibody (Ab-1, Oncogene Science) was also an anti-ER antibody (Ab-1, Oncogene Science) recognizing carried out in these cells. The results are similar to that the C-terminal portion of the ER but was undetectable when observed for the expression of ER (i.e. expression was found anti-ER antibodies recognizing the N-terminal portion of the in tumor tissues but not in cells grown in vitro) (Figure 7), ER (NCL-ER-LH2, Vector laboratory, C314, Santa Cruz) were indicating that the wild type ER expressed in vivo might be used. This observation suggests that the variant ER contains functional. a deletion in the N-terminal region. By RT-PCR analysis using primer pairs in the C-terminal or N-terminal region, the deletion Expression of cell cycle related proteins (p53, p21waf, p16INK4, was found to be in the exon 2 region. Since exon 2 is a part cyclin D1) of DNA binding domain, the ER with deletion in this region The same Western blots studying ER expression (Figure 1) is expected to lose its DNA-binding activity. This was found were also probed for the expression of cell cycle related to be true in the ER-ERE binding assay. That the variant ER proteins. As expected, high levels of p53 were found in SV40 deleting DNA binding domain would be non-functional is transformed cell lines, confirming the involvement of large T revealed by the non-expression of the PgR which is positively antigen in their transformation. The p21 and p16 proteins were regulated by the ER (36). Recently, ER variants with deletion frequently elevated in transformed Type I and Type II HBEC in exon 2, exon 3, or both were observed in normal human cells. The cyclin D1 was highly expressed in Type I normal HBEC compared to their Type II HBEC counterparts. The breast tissue (37). Further RT-PCR analysis will reveal whether transformation by SV40 reduced the expression of cyclin D1 our variant ER also includes deletion in exon 3. in Type I but not Type II HBEC. Of the eight exons that constitute the ER mRNA transcript, most of them (exons 2–7) have been found to be involved in Discussion aberrant splicing events in breast tumor cell lines or tissues (38–42). Many of these variants disrupt critical regions such The major finding of this study is that an estrogen receptor was expressed in all the Type I HBEC with luminal and that they become non-functional (20). The variant ER expressed

Article Snippet: This variant ER appears to determined by a spectrophotometer. contain a deletion in N-terminal region, based on the observa-cDNA was synthesized from the isolated RNA by reverse transcription in tion that, unlike the ER-positive MCF-7 and T47D cells which20 μl reaction solution containing 2.5 μM of random hexamers (Perkin Elmer, Madison, WI), 50 units of Moloney murine leukemia virus reverse transcriptase express both wild type and variant ER, the ER was not (Perkin Elmer, Madison, WI), 1 μg of total RNA, 2 μl of 103 PCR buffer detectable in Type I or Type II HBEC when anti-ER antibodies (500 mM KCl, 100 mM Tris2HCl, pH 8.3), 5 mM MgCl2, 1 mM of each which recognize the N-terminal region (NCL-ER-LH2, Vector dNTP, 20 units of RNase inhibitor (Perkin Elmer, Madison, WI), and 2 μl of Laboratory; C314, Santa Cruz) were used (Figure 1C).RNase free water (Promega, Madison, WI).

Techniques: Western Blot, Expressing, Cell Culture, Variant Assay, Transformation Assay, Injection, In Vitro, Staining, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Plasmid Preparation, In Vivo, Functional Assay, Binding Assay, Activity Assay