Journal: Oncology Letters

Article Title: MicroRNA-744-5p inhibits glioblastoma malignancy by suppressing replication factor C subunit 2

doi: 10.3892/ol.2021.12869

Figure Lengend Snippet: RFC2 expression is upregulated in GBM tissues and cells. (A) RT-qPCR analysis of gene expression of RFC2 in GBM tumor tissues (n=39) and adjacent controls (n=39) from patients with GBM. **P<0.001, compared with Normal group using paired Student's t-test. (B) RT-qPCR analysis of the RFC2 expression in GBM cell lines (U251, U87, SHG44 and A172) and the normal astrocyte NHA cell line. **P<0.001, compared with NHA using one-way ANOVA with Dunnett's test. (C) RT-qPCR analysis of gene expression of RFC2 in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2. (D) Western blot analysis of RFC2 protein expression in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2. (C and D) **P<0.001, compared with CON using one-way ANOVA with Dunnett's test. CON, blank control; si-NC, si-RFC2 negative control; OE-NC, pcDNA 3.1 empty vector; Co-NC, si-NC+OE-NC; si-RFC2, siRNA-RFC2; RFC2-OE, RFC2-overexpression; RFC2, replication factor C subunit 2; GBM, glioblastoma; RT-q, reverse transcription-quantitative; si(RNA), small interfering; OE, overexpression; NC, negative control.

Article Snippet: The human GBM cell lines, U87 (cat. no. HTB-14; GBM of unknown origin) and A172 (cat. no. CRL-1620) were purchased from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Transfection, Western Blot, Control, Negative Control, Plasmid Preparation, Over Expression, Reverse Transcription

Journal: Oncology Letters

Article Title: MicroRNA-744-5p inhibits glioblastoma malignancy by suppressing replication factor C subunit 2

doi: 10.3892/ol.2021.12869

Figure Lengend Snippet: RFC2 promotes cellular proliferation, migration and adhesion, and suppresses cell apoptosis in glioblastoma. (A) Viability of U251 and U87 cells transfected with NC, RFC2-OE and Si-RFC2 was determined by Cell Counting Kit 8 assay. (B) Cellular proliferation was detected in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2 by BrdU assay. (C) Wound-healing assay was performed in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2. (D) Adhesion ability was detected in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2. (E) Caspase3 activity was determined in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2 by caspase3 activity assay kit. (F) Protein expression levels of Bax and Bcl-2 were determined in U251 and U87 cells transfected with NC, RFC2-OE and si-RFC2 by western blotting. *P<0.05 and **P<0.001, compared with CON using one-way ANOVA with Dunnett's test. CON, blank control; NC, negative control; si-RFC2, siRNA-RFC2; RFC2-OE, RFC2-overexpression; RFC2, replication factor C subunit 2.

Article Snippet: The human GBM cell lines, U87 (cat. no. HTB-14; GBM of unknown origin) and A172 (cat. no. CRL-1620) were purchased from the American Type Culture Collection.

Techniques: Migration, Transfection, Cell Counting, BrdU Staining, Wound Healing Assay, Activity Assay, Caspase-3 Activity Assay, Expressing, Western Blot, Control, Negative Control, Over Expression

Journal: Oncology Letters

Article Title: MicroRNA-744-5p inhibits glioblastoma malignancy by suppressing replication factor C subunit 2

doi: 10.3892/ol.2021.12869

Figure Lengend Snippet: RFC2 is a direct target of miR-744-5p in GBM. (A) Bioinformatics analysis showed the predicted binding sequence of RFC2 3′-UTR. (B) Dual luciferase assay was performed in cells co-transfected with WT RFC2 plasmid or MUT RFC2 plasmid and miR-NC or miR-744-5p mimic in U251 and U87 cells. **P<0.001, one-way ANOVA with Tukey's test. (C) Expression of miR-744-5p in GBM tumor tissues (n=39) and adjacent controls (n=39) from patients with GBM was analyzed by RT-qPCR. **P<0.001, compared with Normal group using paired Student's t-test. (D) Correlation between RFC2 and miR-744-5p expression in GBM tissues. (E) RT-qPCR detection of RFC2 expression in NHA, U251 and U87 cells. *P<0.05 and **P<0.001 compared with NHA using one-way ANOVA with Dunnett's test. (F) RT-qPCR analysis of the mRNA expression of RFC2 in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, si-RFC2, and Si-RFC2+ miR-744-5p inhibitor. (G) Western blot analysis of RFC2 protein expression in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, si-RFC2, and si-RFC2+ miR-744-5p inhibitor. (F-G) CON, blank control; NC, negative control. **P<0.001 compared with CON using one-way ANOVA with Dunnett's test. RFC2, replication factor C subunit 2; GBM, glioblastoma; RT-q, reverse transcription-quantitative; si(RNA), small interfering; OE, overexpression; NC, negative control; WT, wild-type; MUT, mutant; miR, microRNA.

Article Snippet: The human GBM cell lines, U87 (cat. no. HTB-14; GBM of unknown origin) and A172 (cat. no. CRL-1620) were purchased from the American Type Culture Collection.

Techniques: Binding Assay, Sequencing, Luciferase, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Negative Control, Reverse Transcription, Over Expression, Mutagenesis

Journal: Oncology Letters

Article Title: MicroRNA-744-5p inhibits glioblastoma malignancy by suppressing replication factor C subunit 2

doi: 10.3892/ol.2021.12869

Figure Lengend Snippet: miR-744-5p targeting to RFC2 suppresses glioblastoma progression. (A) Viability of U251 and U87 cells transfected with NC, miR-744-5p inhibitor, Si-RFC2, and Si-RFC2+ miR-744-5p inhibitor was determined by Cell Counting Kit 8 analysis. (B) Proliferation was detected in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, Si-RFC2, and Si-RFC2+ miR-744-5p inhibitor by BrdU assay. (C) Wound-healing assay was performed in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, Si-RFC2, and Si-RFC2+ miR-744-5p inhibitor. (D) Adhesion ability was detected in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, Si-RFC2, and Si-RFC2+ miR-744-5p inhibitor. (E) Caspase-3 activity was determined in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, Si-RFC2, and Si-RFC2+ miR-744-5p inhibitor by caspase-3 activity assay kit. (F) Protein expression levels of Bax and Bcl-2 were determined in U251 and U87 cells transfected with NC, miR-744-5p inhibitor, Si-RFC2, and Si-RFC2+ miR-744-5p inhibitor by western blot assay. *P<0.05 and **P<0.001, compared with CON using one-way ANOVA with Dunnett's test. CON, blank control; NC, negative control; Si-RFC2, SiRNA-RFC2; Si-RFC2+ miR-744-5p inhibitor, SiRNA-RFC2+ miR-744-5p inhibitor; RFC2, replication factor C subunit 2.

Article Snippet: The human GBM cell lines, U87 (cat. no. HTB-14; GBM of unknown origin) and A172 (cat. no. CRL-1620) were purchased from the American Type Culture Collection.

Techniques: Transfection, Cell Counting, BrdU Staining, Wound Healing Assay, Activity Assay, Caspase-3 Activity Assay, Expressing, Western Blot, Control, Negative Control

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: Characterization of TNTs formed upon treatment with α-SYN protofibrils in astroglia cells (A) The red arrow in the DIC image (a’) indicates TNT formed between two astrocytes on α-SYN protofibril treatment. The bottom panel shows a 3D volume view of Phalloidin and GFAP-positive astrocytes connected by both long (a’’) and short TNTs (b). (B and C) DIC images of U-87 MG cells show TNTs (B) and TMs (C). The cells were stained with phalloidin and β-tubulin; both bottom panels show 3D volume views of TNT as an actin-positive hovering structure (B) and tubulin-positive TM at the surface (C). Red arrows indicate actin-positive TNTs and blue arrows indicate β-tubulin-positive tumor microtubes (TM), respectively. (D and E) Characterization of GJ-negative (D) and -positive (E) TNTs based on Phalloidin and connexin43 staining at the tip of the TNT in U-87 MG cells. Pink arrows indicate close-ended TNT. The bottom panel shows 3D volume views of the same. (F) Closed-ended TM characterized by the presence of connexin43 staining, indicated by pink arrows. The bottom panel shows 3D volume views of the TM at the surface. (G) TNT-like structures (red arrows) are detectable in DIC images as focused structures at z = 4. At z = 0, filopodia-like extensions (magenta arrows) on the substratum are at focus. (H and I) U87MG cells treated with 1μM α-SYN protofibrils for 3h and 6h, stained with lysotracker and MitoTracker, respectively. Red arrows indicate lysotracker and MitoTracker through TNTs at 3h and 6h. Movement of lysotracker (H) and MitoTracker (I) positive vesicles through TNTs were tracked using the TrackMate plug-in of Fiji. (J) Quantification of the percentage of TNTs and (K) percentage of GJ-negative TNTs, GJ-positive TNTs, and TMs from the confocal z-stack images of U-87 MG cells. Quantification of length (L) and diameter (M) of TNTs formed by U-87 MG cells, U251 cells, and astrocytes. Quantifications are done from 10 to 15 image frames of a set and each image frame has 10–20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA (2J and 2K) and one-way ANOVA (2L and 2M). n = 3.

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: Staining

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: TNT biogenesis pathways in cell proliferation (A) Absorbance measured at 570 nm after MTT assay performed in astrocytes treated with varying concentrations (0.5μM, 1μM, 2μM, and 3μM) of α-SYN protofibrils at 3h, 6h, 12h and 24h. (B) Cell number quantification of U-87 MG cells treated with 1μM α-SYN protofibrils after 3h, 6h, 12h, 18h and 24h. (C) MTT absorbance estimated at 24h on treatment with varying concentrations (0.5μM, 1μM, 2μM, and 3μM) of α-SYN protofibrils in U-87 MG cells. (D) Estimation of MTT absorbance at 12h and 24h after treatment with 1μM α-SYN protofibrils in U251 cells. (E) Fluorescence images of astrocytes and U87MG cells treated with 1μM α-SYN protofibrils for 3h–24h were stained with proliferation marker Ki67 along with nuclear stain. (F and G) Quantification of the intensity of Ki67 per cell in astrocytes and U-87 MG respectively. Quantifications are done from 15 image frames of a set and each image frame has 20–25 cells. (H) Flow chart depicting the mode of action of actin inhibitors. (I) DiD (membrane dye) stained, control and 1μM α-SYN protofibrils treated U-87 MG cells pre-treated (before 30min) with 3 μM IPA3, 0.5 μM cytochalasin D, 50 μM CK-666 (Arp2/3 inhibitor), 75 μM blebbistatin and 5 μM Y-27632 (ROCK inhibitor). Red arrows indicate the formation of TNT-like structures observed at 3 h and (K) shows the quantification of TNT numbers. (J) representative images showing cell numbers with the above-mentioned treatment with inhibitors and α-SYN protofibrils at 24 h. (L and M) quantification of MTT absorbance and cell numbers of the same, respectively. Quantifications are done from 5 image frames of a set and each image frame has 15–20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA, and only graph 6B was analyzed using one-way ANOVA. n = 3.

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: MTT Assay, Fluorescence, Staining, Marker, Membrane, Control

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet: FAK translocation, ROCK remodulation, and activation of proliferation pathway (A) Estimation of uptake of α-SYN-TMR protofibrils by U251 cells treated with 1μM α-SYN protofibrils and pre-treated (before 30min) with 0.5 μM cytochalasin D and 5 μM Y-27632 (ROCK inhibitor) by flow cytometry. (B) Quantification of percentage gated fluorescence intensity of α-SYN-TMR protofibrils in the above experiment. (C) Fluorescence images of maximum intensity projected confocal z-stacks in astrocytes stained with Phalloidin (green) and FAK (red). The upper panel has nucleus stained with DAPI and the lower panel is without DAPI. White arrows indicate the nuclear colocalization of FAK, red arrows indicate TNTs and pink arrows indicate FAK at focal adhesion. (D) Quantification of FAK intensity in the nucleus of the cells with and without TNTs. (E and G) Fluorescence images of maximum intensity projected confocal z-stacks in U-87 MG cells stained with FAK (red) and pFAK (red) with DAPI, respectively. (F and H) Quantification of FAK and pFAK fluorescence intensity per cell in the nucleus. Quantifications are done from 15 image frames in a set, and each image frame has 15–20 cells. (I) Western blot images showing the change in ROCK1 and ROCK2 expressions with time on 1μM α-SYN protofibril treatment in U-87 MG cells. (J) Quantification of ROCK1 and ROCK2 western blots. Full-length western blots of four repeats are represented in Figure S9 . (K) Western blot images showing increased intensity of pERK1/pERK2, NF-κB, and Cdk1 with time on 1μM α-SYN protofibril treatment in U-87 MG cells. Full-length western blots of three repeats for pERK1/pERK2 and NF-κB and five repeats for Cdk1 were represented in Figure S10 . (L) Quantification of pERK1/pERK2, NF-κB, and Cdk1 western blots. Scale bars are denoted on the images. Data are expressed as mean ± SD, ∗∗∗ p ≤ 0.001. Statistics were analyzed using a two-way ANOVA. n = 3.

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: Translocation Assay, Activation Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: iScience

Article Title: Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

doi: 10.1016/j.isci.2024.110565

Figure Lengend Snippet:

Article Snippet: U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India (Original source; European Collection of Authenticated Cell Cultures, ECACC).

Techniques: Recombinant, Staining, Software