Journal: Scientific Reports

Article Title: The oncogenic role of TIMM8A in cancer and the mechanistic insights into the function in breast cancer cells

doi: 10.1038/s41598-025-03331-x

Figure Lengend Snippet: TIMM8A knockdown inhibited the migration and invasion of breast cancer in vitro. ( A – C ) Migration capacity of MCF7 and MDA-MB-231 cells after knockdown of TIMM8A assayed by scratch experiment. Area Recovery (%) = (initial scratch area—final scratch area)/initial scratch area × 100%. ( D – F ) Migration capacity of MCF7 and MDA-MB-231 cells after knockdown of TIMM8A assayed by transwell migration assay. ( G – I ) Invasion capacity of MCF7 and MDA-MB-231 cells after knockdown of TIMM8A assayed by transwell invasion assay. ( J – R ) The expression levels of NF-κB p65 and EMT-related proteins, including α-SMA, Vimentin, E-cadherin and N-cadherin in MCF7 and MDA-MB-231 cells after knockdown of TIMM8A. GAPDH was used as a loading control. The results shown are representative of at least three independent experiments. EMT, epithelial-mesenchymal transition. The student’s t-test or one-way ANOVA was used to detect the differences among groups. * P < 0.05, ** P < 0.01, *** P < 0.001. Samples were derived from the same experiment and gels/blots were processed in parallel. Original blots/gels are presented in the Supplementary File.

Article Snippet: The fundamental procedures were as follows: the transwell chamber (invasion assay: Matrigel® invasion chamber 8.0micron, Corning, USA; migration assay: 24 well multiwell insert System 8.0micron, Falcon, USA) is placed in the 24-well plate, the chamber is called the upper chamber and the plate is called the lower chamber.

Techniques: Knockdown, Migration, In Vitro, Transwell Migration Assay, Transwell Invasion Assay, Expressing, Control, Derivative Assay

Journal: Cell reports

Article Title: MIRO2 promotes cancer invasion and metastasis via MYO9B suppression of RhoA activity

doi: 10.1016/j.celrep.2024.115120

Figure Lengend Snippet: The indicated cell lines were transfected with control (C) or MIRO2-targeting siRNA (M2a and M2b, two independent sequences) and used for downstream assays at 72 h post-transfection. (A) Western blot was carried out to confirm MIRO2 knockdown. Representative blots from n = 3 experiments are shown. (B and C) Cells were seeded in transwell invasion assays and allowed to invade for 16–24 h. (B) Representative images of invasive cells stained with DAPI. (C) Quantification of invaded cells/field, relative to control. Data are represented as the mean ± SEM ( n = 3), and means were compared by one-way ANOVA and Dunnett’s post-test. (D) Cell growth was determined by CyQUANT cell proliferation assay measured at the time of plating and at 24 h post-plating. Data were calculated as the differential growth relative to control and are represented as the mean ± SEM ( n = 3). Means were compared by one-way ANOVA and Dunnett’s post-test. (E) Comparison of the changes in invasion and growth in MIRO2 knockdown cells relative to control cells from (C) and (D), respectively. Data are represented as the mean ± SEM ( n = 3), and means were compared by two-tailed unpaired t test. For (C)–(E), p values are represented as ns, not significant ( p > 0.05), or * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001. See also .

Article Snippet: Invasion experiments were performed by plating 5×10 4 –1.5×10 5 cells in duplicate onto growth factor reduced Matrigel coated invasion transwell chambers (Corning, catalog# 08–774-193).

Techniques: Transfection, Control, Western Blot, Knockdown, Staining, CyQUANT Assay, Proliferation Assay, Comparison, Two Tailed Test

Journal: Cell reports

Article Title: MIRO2 promotes cancer invasion and metastasis via MYO9B suppression of RhoA activity

doi: 10.1016/j.celrep.2024.115120

Figure Lengend Snippet: The indicated cell lines were transfected with control (C) or MYO9B-targeting siRNA (M9B-a and M9B-b, two independent sequences) and used for downstream assays at 72 h post-transfection. (A) Western blot was carried out to confirm MIRO2 knockdown. Representative blots from n = 3 experiments are shown. (B) Cells were seeded in transwell invasion assays and allowed to invade for 18–24 h. Representative images of invasive cells stained with DAPI are provided. (C) Quantification of invasive cells/field relative to control. Data are represented as the mean ± SEM ( n = 3), and means were compared by one way ANOVA and Dunnett’s post-test. (D) Cell growth was determined by CyQUANT cell proliferation assay measured at the time of plating and at 24 h post-plating. Data were calculated as the differential growth relative to control. Data are represented as the mean ± SEM ( n = 3), and means were compared by one-way ANOVA and Dunnett’s post-test. (E) Comparison of the changes in invasion and growth in MYO9B-knockdown cells relative to control cells from (C) and (D), respectively. Data are represented the mean ± SEM ( n = 3), and means were compared by two-tailed unpaired t test. For (C)–(E), p values are represented as ns, not significant ( p > 0.05), or * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001. See also .

Article Snippet: Invasion experiments were performed by plating 5×10 4 –1.5×10 5 cells in duplicate onto growth factor reduced Matrigel coated invasion transwell chambers (Corning, catalog# 08–774-193).

Techniques: Transfection, Control, Western Blot, Knockdown, Staining, CyQUANT Assay, Proliferation Assay, Comparison, Two Tailed Test

Journal: Cell reports

Article Title: MIRO2 promotes cancer invasion and metastasis via MYO9B suppression of RhoA activity

doi: 10.1016/j.celrep.2024.115120

Figure Lengend Snippet: (A) MDA-MB-231 cells were transfected with control, MYO9B (M9B), or MIRO2 (M2) pooled siRNA in combination with a Rho sensor (dTomato-2xrGBD), plated into collagen-coated slides, and analyzed via fluorescence microscopy. Arrows point to ruffles selected for analysis and zoomed panels include region of line scans. (B) Quantification of Rho sensor signal enrichment at the ruffles. Data are represented as the mean ± SEM, and means were compared by one-way ANOVA and Dunnett’s post-test. (C–E) The indicated cell lines were transfected with control, MIRO2 (M2), RhoA (RA), or a combination of both MIRO2 and RhoA (M2/RA) pooled siRNA and used for downstream assays at 72 h post-transfection. (C) Representative blots showing the efficiency of knockdown. (D) Cells were seeded in transwell invasion chambers and allowed to invade for 18–24 h. Representative images of invasive cells stained with DAPI are shown. (E) Quantification of invasive cells/field, relative to control. Data are represented as the mean ± SEM ( n = 4 for MDA-MB-231 and n = 5 for PC3), and means were compared by one-way ANOVA and Dunnett’s post-test. (F–H) The indicated cell lines were transfected with either control or MIRO2 (M2) pooled siRNA for 24 h, followed by cDNA transfection of either EGFP-empty (EV), EGFP-MYO9B-wildtype (WT), or EGFP-MYO9B-R1695M (GAP deficient, GD) for 48 h. (F) Representative western blots at time of plating. (G) Cells were seeded in transwell invasion chambers and allowed to invade for 18–24 h. Representative images of invasive cells stained with DAPI are shown. (H) Quantification of invasive cells/field, relative to control. Data are represented as the mean ± SEM ( n = 3), and means were compared by one-way ANOVA and Dunnett’s post-test. For (B), (E), and (H), p values are represented as ns, not significant ( p > 0.05), or * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001. See also and .

Article Snippet: Invasion experiments were performed by plating 5×10 4 –1.5×10 5 cells in duplicate onto growth factor reduced Matrigel coated invasion transwell chambers (Corning, catalog# 08–774-193).

Techniques: Transfection, Control, Fluorescence, Microscopy, Knockdown, Staining, Western Blot