Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls ploidy in Y235T cells. A The presented bar graph illustrates the GJB3 mRNA amounts across various human tissues, with detailed information available in the Materials and Methods section. Urothelial cells (UC#1 and UC#2) were isolated from ureters from two separate patients who underwent nephrectomy at Ulm University. The mRNA levels were normalized to GAPDH . n = 3 independent experiments were performed. Error bars represent mean ± SEM. B The representative pictures display the HE, GJB3 and IgG staining in human ureter tissues (U#1 and U#2, respectively). C The representative Western blot result indicates GJB3 protein levels in Y235T cells with shGJB3. α-tubulin is used as a loading control. n = 3 independent experiments were performed. D The bar graphs depict the effectiveness of GJB3 knockdowns at the mRNA level in Y235T cells, with the measurements reference to the GAPDH mRNA level. n = 3 independent experiments were performed. Error bars represent mean ± SEM. E Representative pictures showing metaphase spreads of Y235T cells with shControl and shGJB3#2. Chromosomes are visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Control cells showing 46 chromosomes in most metaphase spreads. Exemplary pictures demonstrating the induction of aneuploidy in Y235T cells subsequent to GJB3 knockdown. The images show a metaphase spread of Y235T-shGJB3#2 cells with 51 chromosomes. F Chromosomes numbers of metaphase spreads from Y235T cells that were knockdown GJB3. n = numbers of (Each counting is indicated within the graph). Results are pooled from three independent sets of experiments. Mean ± SEM values are shown in the dot plot, and significance was determined by using Fisher’s exact test. G Representative pictures showing micronuclei of Y235T cells with shGJB3#1. Cell nuclei are stained with DAPI, and phalloidin Alexa Fluor 488 was used for F-actin visualization. White arrows indicate micronuclei. H Quantitation of cells with micronuclei upon knockdown of GJB3. Results from n = 3 separate series of experiments. The bar graph displays the mean ± SEM values, and the two-tailed Student's t-test was used to assess the significance. I Immunofluorescence results indicate the multinucleation of Y235T shGJB3#1 cell. Cell nuclei is visualized by DAPI, and F-actin is visualized by Alexa Fluor 488. J Quantitation of cells with multinucleation with knockdown of GJB3. Results from n = 3 independent sets of experiments. Mean ± SEM values are shown in the bar graph, and the significance was determined by two-tailed Student’s t -test. K Figures depict of mitotic abnormalities in metaphase and anaphase. DAPI (blue) indicates chromosomes, Cy5 (red) indicates α-tubulin, and Alexa Fluor 488 (green) labeling illustrates γ-tubulin. White arrows are used to indicate chromatid mislocation or multipolar centrosomes. L – M Quantitative evaluation of mitotic abnormalities. Results from n = 3 distinct experiments. The bar graph displays mean ± SEM data, and a two-tailed Student’s t -test was used to assess significance. Scale bars: 200 μm ( B main panels) 50 μm ( B insets) 20 μm ( E , G , I ) and 2 μm ( K ). Images are shot at total magnification of 100x ( B main panels), 630x ( B insets, E , G , I , K )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Isolation, Staining, Western Blot, Control, Knockdown, Quantitation Assay, Two Tailed Test, Immunofluorescence, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 controls spindle orientation and microtubule dynamics. A Exemplary pictures illustrating the disorientation of Y235T-shGJB3#1 cells. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. B Quantitative assessment of the spindle pole displacement factor (SPDF) in Y235T cells. n = 256 (Y235T-shScr), 253 (Y235T-shGJB3#1), 263 (Y235T-shGJB3#2), C , Representative images showing reorientation of UMUC3 cells with ectopic GJB3. The chromosomes are indicated by DAPI, γ-tubulin is visualized by Alexa Fluor 488, and α-tubulin is visualized by Cy5. D Quantitative assessment of the spindle pole displacement factor (SPDF) in UMUC3 cells. n = 155 (UMUC3-EV), and 140 (UMUC3-GJB3). Experiments present combined data from three separate sets of independent experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. To boost the proportion of prometaphase cells, cells were treated to dimerthylenastron for four hours prior to labeling. E Examples of images demonstrating microtubule growth in Y235T cells expressing shGJB3#2. F Rates of mitotic microtubule plus-end assembly in Y235T-shGJB3 cells. n = 60 cells are pooled from three independent sets of experiments. G Example of images demonstrating growth of microtubules in UMUC3-GJB3 cells. H Rates of mitotic microtubule plus-end assembly in UMUC3-GJB3 cells. n = 60 cells are combined from three separate sets of experiments. The two-tailed Student’s t -test was used to evaluate significance, and the mean ± SEM data are displayed in the dot plot. GJB3 interacts with α-tubulin. The deletion of GJB3 in UROtsa cells using the CRISPR-cas9 method was detailed in the main article. I Western blot displaying GJB3 protein levels in UROtsa cells with a control guide RNAs directed against green flourescent protein or two distinct gRNAs targeting GJB3 (gGJB3#1 and GJB3#2). α-tubulin serves as loading control. n = 3 separate experiments were conducted. J Exemplary pictures displaying GJB3 and α-tubulin colocalization in UROtsa cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. K Quantitation of GJB3 and α-tubulin colocalization in UROtsa cells by Pearson’s correlation coefficient. n = 49 (UROtsa-gControl), 58 (UROtsa-gGJB3#1), 53(UROtsa-gGJB3#2) are pooled from three to four independent experiments. L GJB3 protein level in UMUC3 cells with ectopic GJB3 was detected by Western blot. α-tubulin is used as a loading control. n = 3 independent experiments were performed. M Representative images displaying the colocalization of GJB3 and α-tubulin in UMUC3 cells during metaphase. GJB3 is visualized by Alexa Fluor 488 and α-tubulin is visualized by Cy5. Yellow signal indicates the overlap of GJB3 and α-tubulin. N Quantitation of GJB3 and α-tubulin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 105 (UMUC3-EV), and 64 (UMUC3-GJB3) are pooled from three to four independent experiments. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t-test ( M , N ). O – P GJB3 bundle microtubule (MT) filament level was detected by Western blot. 5 × 10 11 MT/ml and 5–10 μm in length MTs were incubated with increasing concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . ( O ), Flag-GJB3, indicated by red arrowheads and ( P ), microtubules, indicated by red arrows, are visualized by specific antibodies. n = 3 independent experiments were performed. Scale bars: 5 μm ( A , C) and 1 μm ( E , G) and 2 μm ( J , M ). Images were captured at total magnification of 630x

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Two Tailed Test, Labeling, Expressing, CRISPR, Western Blot, Control, Quantitation Assay, Incubation, SDS Page, Centrifugation

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 as tumor suppressors in bladder cancer. A–B ( A ) RT-qPCR was used to measure the GJB3 mRNA expression , and ( B ) Western blot was employed to ascertain protein amounts. The relative mRNA expression was quantified with respect to GAPDH . The qPCR shows results of n = 4 technical repeats. Error bars represent Mean ± SEM. α-tubulin serves as loading control in Western blot. n = 3 independent experiments were performed. C Comparison of GJB3 mRNA levels in the CNUH (GSE13507) cohort of NMIBC ( n = 103) and MIBC ( n = 62) human bladder tumors. Statistical differences were defined by two-way Fisher’s ANOVA test * = p ≤ 0.05. D The IHC images represent the GJB3 staining (red arrows) in human normal bladder and bladder cancer tissues. E Quantitation of GJB3-IHC staining scores in human normal bladder and bladder cancer groups. F The IHC images display Gjb3 staining (red arrows) in mouse normal bladder and bladder cancer tissues during BBN-induced BC progression. G Quantitation of Gjb3-IHC staining scores in normal bladder and bladder cancer tissues during the BBN-induced BC progression in mice. The expression of Gjb3 gradually diminished after the BBN treatment, in contrast to the control mice (black dots), which were given water (orange dots). The bar graph displays mean ± SEM data, and a two-tailed Student's t-test was used to assess significance. Scale bars: 200 μm ( D and F , main panels) and 50 μm ( D and F insets). Images were captured at total magnification of 100 × ( D and F main panels), and 630 × ( D and F insets)

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Comparison, Staining, Quantitation Assay, Immunohistochemistry, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 inhibits cells invasion and migration. A – B Western blots displaying the GJB3 protein quantity assessments in RT4 and in UMUC3 cells with experimental modifications. The loading control was provided by α-tubulin levels. The Western blots were repeated for three times. C Cell migratory capacity in RT4 cell line with shGJB3#1 was detected by Wound healing/scratch. The exemplary imaged were captured at 24 and 120 h. D Normalized cell free area was used to quantify the impact of GJB3 knockdown on RT4 cells by Wound healing assay. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. E Cell migratory capacity in UMUC3 cell line with ectopic GJB3 was detected by Wound healing/scratch. The representative pictures captured at 12 and 24 h in case of UMUC3 cells. F Quantitation of normalized cell free area of UMUC3 cells with ectopic GJB3 performed by Wound healing assay n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. G The invasion capacity of RT4 cell line with shGJB3#1 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 144 h post-seeding. H Quantitation of invasive capacity of RT4 cells expressing the indicated shRNAs targeting GJB3. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student’s t -test was used to assess significance. I The invasion capacity of UMUC3 cell line with ectopic of GJB3 was detected by Boyden chamber. The exemplary images depict cell invasion through the Boyden chamber, stained at 48 h post-seeding. J, Quantitation of invasive capacity of UMUC3 cells with ectopic GJB3 expression. n = 3 distinct experiments. The bar graphs display mean ± SEM values, and a two-tailed Student's t-test was used to assess significance. K Representative images of hematoxylin/eosin stainings showing the invasion capacity of RT4 cell line with shGJB3#2 by porcine bladder ex vivo organ culture method (the invasive capacity of BC cells in the ex vivo organ culture model was quantified as shown in Fig. S4). The cells were seeded on the surface of the de-epithelized porcine bladder for 21 days. L Quantitation graphs displaying the impact of GJB3 alteration on the invasive capacity of RT4 cells in ex vivo organ culture approach. n = 4 (RT4-shScr), n = 3 (RT4-shGJB3#1), n = 3 (RT4-shGJB3#2) M Representative images of hematoxylin/eosin stainings showing the invasion capacity of UMUC3 cell line with ectopic GJB3 by porcine bladder ex vivo organ culture approach. The cells were seeded on the surface of the de-epithelized porcine bladder for or 14 days. Insets: enlarged images of the areas shown by black boxes. Black arrows indicate the cells that have spread the farthest from the surface. n = 3 (UMUC3-EV), n = 4 (UMUC3-GJB3) independent experiments were performed. Mean ± SEM values are shown in the bar graph, and significance was determined by two-tailed Student’s t -test. Scale bars: 200 μm ( C , E , G , I , K main panels, M main panels) and 100 μm ( K insets, M insets). Images were captured at total magnification of 50 × ( C , E ), 100 × ( G , I , K main panels, M main panels), and 200 × ( K insets, M insets). O - V Morphological changes and actin-enriched protrusions in UMUC3, and RT4 cells with altered GJB3 expression. Cells with actin-enriched protrusions are marked with white arrows. O - P Brightfield microscopy images depict cells exhibiting a transition towards a round morphology ( O ) of UMUC3 cells with ectopic GJB3 expression. P RT4 cells with GJB3 knockdown demonstrate an elongated shape. Magnifications indicate 100 × or 400 × , respectively. The scale bars refer to 100 μm (left), and 50 µm (right). Q - T Quantification of round or elongated morphology on fixed cells. A cell with elongated or round morphology is identified by the ratio of longest and shortest diameter of the cell from images captured randomly at 630 × magnification using a Zeiss TCS SP5 confocal microscope. Scale bars: 20 μm. The ratio is calculated as the longest diameter of the cell dividing by the shortest diameter of the cell. The ratio is calculated as longest diameter dividing by shortest diameter. A cells with Ratio ≤ 2 is identified as round morphology, while ratio > 2 is elongated morphology. Q , R Immunofluorescence staining photos illustrate the round morphology shifting of ( Q ) UMUC3 cells with GJB3 overexpression compared to cells transfected with empty vector (EV). R RT4 cells with GJB3 knockdown display a transition towards an elongated morphology compared to cells transfected with shScramble (shScr). Cell nuclei are stained with DAPI, and F-actin is labeled with Phalloidin-AF488. S , T The bar graphs reveals percentages of cells with different morphology in total in each group, shown above in Q and R . The percentage was calculated as number of cells with elongated (or rounded) morphology divide cell numbers in total. The percentage values in different groups are marked above or in the bars. Black bars indicate percentages of cells with elongated morphology, and gray bars indicate the percentage of cells with rounded morphology. The statistical significance is calculated by using chi-square statistic. U , V The graphs show the fraction of cells with actin-enriched protrusions in response to GJB3 alterations ( U ) in RT4 or ( V ) in UMUC3 cells. For each picture, the percentage of cells with actin-enriched protrusions is calculated by the number of the cells with actin-enriched protrusions divided by total number of cells. (n) indicates the number of pictures taken in the group

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Migration, Western Blot, Control, Knockdown, Wound Healing Assay, Two Tailed Test, Quantitation Assay, Staining, Expressing, Ex Vivo, Organ Culture, Microscopy, Immunofluorescence, Over Expression, Transfection, Plasmid Preparation, Labeling

Journal: Cellular & Molecular Biology Letters

Article Title: Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion

doi: 10.1186/s11658-024-00609-2

Figure Lengend Snippet: GJB3 interactics with F-actin and influences invadopodia formation via actin dynamics. A Representative images demonstrating invadopodia formation of RT4 cells with shGJB3#2. B Quantitation of the invadopodia number was performed in RT4 cells. n = 959 (RT4-shScr), n = 718 (RT4-shGJB3#1), n = 481 (RT4-shGJB3#2). Cells are pooled from three independent sets of experiments. C, Representative pictures showing invadopodia formation of UMUC3 cells upon ectopic GJB3 expression. D Quantitation of the invadopodia number was performed in UMUC3 cells. n = 110 (UMUC3-EV), n = 137 (UMUC3-GJB3). Cells are pooled from three independent sets of experiments. F-actin is visualized by Alexa Fluor 488-phalloidin and Cortactin is visualized by Cy5, respectively. Yellow spots displaying cortactin and F-actin colocalization identify the invadopodia structures. The regions indicated by white boxes are magnified in the insets. The invadopodia are marked with white arrows. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. E Representative pictures indicate the gelatin degradation by RT4 cells upon GJB3 knockdown. F Gelatin degradation capacity of the cells was quantified by measuring the degradation area per RT4 cell. n = 2809 (RT4-shScr), n = 2447 (RT4-shGJB3#1), n = 3544 (RT4-shGJB3#2). G Representative pictures indicate the gelatin degradation by UMUC3-GJB3 cells. H Gelatin degradation capacity of the cells was quantified by measuring the degradation area per UMUC3 cell. n = 1048 (UMUC3-EV), and n = 1246 (UMUC3-GJB3) are pooled from three to four independent experiments. The dot plot displays the mean ± SEM data, and the two-tailed Student's t-test was used to assess significance. I The LifeAct–Ruby signal recovery duration in UMUC3 cells with or without GJB3 is depicted in representative images after LifeAct–Ruby signal bleaching. The white arrows indicate the areas of bleaching. J , The quantitation of bleaching recovery experiments with UMUC3 cells. n = 29 (UMUC3-EV), n = 31 (UMUC3-GJB3). Three different groups of separate experiments' cells are combined. The graphs' data points correspond to the mean ± SEM. P values were calculated using the two-tailed Student's t -test at t = 49 s. K Exemplary pictures displaying the colocalization of GJB3 with F-actin in control UROtsa cells. Insets: enlarged image of the areas shown by white box. L , Quantitation of GJB3 and F-actin colocalization in UROtsa by Pearson’s correlation coefficient. n = 161 (UROtsa-gControl), n = 206 (UROtsa-gGJB3#1), n = 276 (UROtsa-gGJB3#2). M Exemplary pictures displaying the GJB3/F-actin colocalization in UMUC3-GJB3 cells. Insets: enlarged image of the areas shown by white box. N Quantitation of GJB3 and F-actin colocalization in UMUC3 cells by Pearson’s correlation coefficient. n = 672 (UMUC3-EV), and n = 831 (UMUC3-GJB3) are combined from 3 separate experiments. The two-tailed Student's t-test was used to establish significance, and the bar graph displays mean ± SEM results. Alexa Fluor 647 illustrates the F-actin, and Alexa Fluor 488 illustrates GJB3. Yellow highlights denote GJB3 and F-actin overlap. Insets: enlarged images of the colocalized areas shown by white boxes. O , P GJB3 binds bundle actin filaments in a dose-dependent manner by Western blot. Actin (2.5 mg/ml) concentrations of GJB3 (relative GJB3 amount is indicated by + or + +). Supernatant (S) and pellet (P) were subjected to 10% SDS-PAGE after high-speed centrifugation at 100,000 g . Red arrowheads indicate the GJB3, and the red arrow indicates actin filaments visualized by western blot with specific antibodies. n = 3 independent experiments were performed. Scale bars: 10 μm ( A , C , E , G , K and M main panels), 1 μm ( A , C insets), 2 μm ( E , G , K and M insets) and 1 μm ( I ). Images were captured at total magnification of 630 × ( A , C , I , K , M ) and 400 × ( E , G )

Article Snippet: The following primary antibodies were utilized: Anti-GJB3 rabbit antibody (1:2000 for Western blot (WB) and 1:200 for immunofluorescence (IF), ab236620, Abcam, Cambridge, UK); Anti-GJB3 mouse antibody (1:500 for WB and 1:200 for IHC on mouse samples; 1:400 for immunohistochemistry (IHC) on human samples, sc-81803, Santa Cruz, California, USA); Anti-Flag rabbit antibody (1:2000 for WB, F7425, Sigma-Aldrich, St. Louis, USA); Anti-α-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5168, Sigma-Aldrich, St. Louis, USA); Anti-Cortactin mouse antibody (1:500 for IF, #H5, Santa Cruz, California, USA); Anti-γ-tubulin mouse antibody (1:2000 for WB and 1:500 for IF, T5192, Sigma-Aldrich, St. Louis, USA); Anti-β-actin mouse antibody (1:10,000 for WB, A1978, Sigma-Aldrich, St. Louis, USA).

Techniques: Quantitation Assay, Expressing, Two Tailed Test, Knockdown, Control, Western Blot, SDS Page, Centrifugation

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging