Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) Western blot showing expression changes of several N-cad binding catenins following 6–12 cycles of irradiation (5 Gy) in mGS cells. (B) Fluorescence microscopy shows that β-catenin (green) selectively coaccumulates with N-cad (red) on the cell surface of mGSRR but not mGS cells. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 25 μm. (C) Wnt/β-catenin regulated transcriptional activity in mGS and mGSRR cells measured through transient transfection with a luciferase reporter driven by a WT (TOP) or mutant (FOP) TCF binding site. ***P < 0.001, 2-tailed Student’s t test. (D) TOP/FOP ratio showing Wnt/β-catenin activity in parental N-cad–overexpressing and N-cad–KO mGS cells. **P < 0.01, ***P < 0.001, Tukey’s HSD test. (E) Microarray analysis showing that mRNA expression of multiple Wnt target genes is suppressed in mGSRR compared with mGS cells. Each group contains 2 independent replicates (n = 2). (F) qRT/PCR showing that NeuroD1, Ngn1, and Brn3a mRNAs are reduced in mGSRR cells. Two-tailed Student’s t test. (G) Western blot showing expression change of β-catenin (pan and non-phospho), c-Myc, and Tuj1 by N-cad–overexpressing and N-cad–KO mGS cells. All blots show representative images (n = 3 or more).

Article Snippet: Luciferase reporter gene transfections were performed with lipofectamine using a reporter gene construct driven by a TCF binding site (M50 Super 8x TOP Flash, Addgene, #12456) and a negative control with a mutant TCF binding site (M51 Super 8x FOP Flash, Addgene, #12457), which were provided by Randall Moon (University of Washington, Seattle, Washington, USA).

Techniques: Western Blot, Expressing, Binding Assay, Irradiation, Fluorescence, Microscopy, Activity Assay, Transfection, Luciferase, Mutagenesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Convergence of autism proteins at the cilium

doi: 10.1101/2024.12.05.626924

Figure Lengend Snippet: (A) Human GFP-tagged constructs for autism-associated transcription factors SATB2 and RFX3 (green) localize to the nucleus (inset, deeper z-plane) and do not localize to motile cilia, while GFP-tagged constructs for autism-associated chromatin regulators ADNP, CHD8, CHD2, and POGZ do localize to motile cilia (labeled by acetylated alpha-Tubulin, magenta; basal bodies labeled by Centrin-CFP, cyan) when expressed in the X. laevis embryonic epidermis. Bottom panel shows basal bodies, actin, and autism-associated proteins in greater detail (actin network labeled by phalloidin, gray). (B) Human GFP-tagged constructs for POGZ, CHD8, and CHD2 (green) localize to primary cilia (cilia marked by Flag-tagged D1 receptor, magenta; basal bodies labeled by FGFR1OP antibody, red) when expressed in primary rat striatal neurons. (C) Endogenous staining for POGZ, CHD8, and CHD2 (green) in primary cilia (labeled by ARL13B, magenta; basal bodies labeled by FGFR1OP, red) in primary rat striatal neurons. (D) Morpholino-mediated knockdown (KD) of chd8 , chd2 , or pogz in X. tropicalis results in defects in cilia (labeled by acetylated alpha tubulin, magenta) on multiciliated cells of the embryonic epidermis. (E) Quantification of the data shown in B, with cilia defects categorized as “normal,” “moderate,” or “severe”. See also - and Table S3.

Article Snippet: Primary antibodies used: acetylated alpha-Tubulin (Sigma T6793, 1:600 for Xenopus , 1:1000 for rat brain slices), ARL13B (BiCell Scientific 90413h, 1:100 for human iPSC-derived neurons), CHD8 (Abcam ab114126, 1:100 for Xenopus and for rat striatal neurons), CHD2 (Novus Biologicals NBP2-32563, 1:100 for Xenopus and for rat striatal neurons), POGZ (Abcam ab167408, 1:100 for Xenopus and for rat striatal neurons), SYNGAP1 (MyBioSource MBS3216405, 1:100 for Xenopus ), SYNGAP1 (Abcam ab3344, 1:1000 for rat ependymal cells or human iPSC-derived neurons), SYNGAP1 (Invitrogen PA1-046, 1:500 for rat striatal neurons), STXBP1 (Synaptic Systems 116003, 1:500 for rat striatal neurons, 1:150 for rat ependymal cells), SHANK3 (Santa Cruz Biotechnology, sc-377088, 1:1000 for rat ependymal cells), PSD95 (Santa Cruz Biotechnology, sc-32290, 1:200 for rat striatal neurons, 10% donkey serum block), SCN2A (Alomone labs ASC-002, 1:100 for rat striatal neurons), SLC6A1 (Novus Biologicals NBP1-89802, 1:100 for rat striatal neurons), GRIN2B (Abcam ab65783, 1:200 for rat striatal neurons), FGFR1OP (Abnova H00011116-M01, 1:500), FGFR1OP (ProteinTech 11343-1-AP, 1:100), ARL13B-647 (ProteinTech CL647-17711, 1:500), and anti-GFP (Novus NB100-1614, 1:1000).

Techniques: Construct, Labeling, Staining, Knockdown

Journal: bioRxiv

Article Title: Convergence of autism proteins at the cilium

doi: 10.1101/2024.12.05.626924

Figure Lengend Snippet: (A) Human GFP-tagged constructs for autism-associated ‘neuronal communication’ proteins (green) known to localize across three different neuronal subcompartments—presynaptic density, axon initial segment (AIS), and the postsynaptic density—localize to cilia (axonemes labeled by acetylated alpha-Tubulin, magenta; basal bodies labeled by centrin-CFP, cyan) when expressed in X. laevis . NRXN1 localizes to the actin network surrounding cilia (labeled by phalloidin, gray). Bottom panel shows basal bodies and autism-associated proteins in greater detail. H2B-GFP control localizes to the nucleus, in a deeper Z-plane as indicated in the inset labeled “Nuclear Z-plane.” (B) Human GFP-tagged constructs for SLC6A1, SYNGAP1, PSD95, and SCN2A (green) localize to primary cilia (cilia labeled by Flag-tagged Dopamine D1 receptor, magenta; basal bodies labeled by FGFR1OP, red) when expressed in primary rat striatal neurons. (C) Endogenous staining for SLC6A1, SYNGAP1, PSD95, and SCN2A in primary cilia (labeled by ARL13B, magenta; basal bodies labeled by FGFR1OP, red) in primary rat striatal neurons. (D,E) Loss of syngap1 , via CRISPR ( syngap1 CR ) or morpholino-mediated knockdown ( syngap1 KD ) , results in cilia defects (labeled by acetylated alpha-Tubulin, magenta) and disrupts the apical actin network (labeled by phalloidin, gray) in the X. tropicalis embryonic multiciliated epidermis. (F) Quantification of the syngap1 KD displayed in E as a percentage of embryos with no cilia phenotype vs. with a moderate or severe cilia phenotype. Fisher’s exact test (two-sided) followed by Pairwise Fisher’s Exact tests were used to calculate significance using raw counts of individual embryos scored as having no cilia phenotype or moderate to severe cilia phenotypes. ** = p < 0.01, ns = no statistically significant difference. See also - and Tables S3 and S5.

Article Snippet: Primary antibodies used: acetylated alpha-Tubulin (Sigma T6793, 1:600 for Xenopus , 1:1000 for rat brain slices), ARL13B (BiCell Scientific 90413h, 1:100 for human iPSC-derived neurons), CHD8 (Abcam ab114126, 1:100 for Xenopus and for rat striatal neurons), CHD2 (Novus Biologicals NBP2-32563, 1:100 for Xenopus and for rat striatal neurons), POGZ (Abcam ab167408, 1:100 for Xenopus and for rat striatal neurons), SYNGAP1 (MyBioSource MBS3216405, 1:100 for Xenopus ), SYNGAP1 (Abcam ab3344, 1:1000 for rat ependymal cells or human iPSC-derived neurons), SYNGAP1 (Invitrogen PA1-046, 1:500 for rat striatal neurons), STXBP1 (Synaptic Systems 116003, 1:500 for rat striatal neurons, 1:150 for rat ependymal cells), SHANK3 (Santa Cruz Biotechnology, sc-377088, 1:1000 for rat ependymal cells), PSD95 (Santa Cruz Biotechnology, sc-32290, 1:200 for rat striatal neurons, 10% donkey serum block), SCN2A (Alomone labs ASC-002, 1:100 for rat striatal neurons), SLC6A1 (Novus Biologicals NBP1-89802, 1:100 for rat striatal neurons), GRIN2B (Abcam ab65783, 1:200 for rat striatal neurons), FGFR1OP (Abnova H00011116-M01, 1:500), FGFR1OP (ProteinTech 11343-1-AP, 1:100), ARL13B-647 (ProteinTech CL647-17711, 1:500), and anti-GFP (Novus NB100-1614, 1:1000).

Techniques: Construct, Labeling, Control, Staining, CRISPR, Knockdown

Journal: bioRxiv

Article Title: Convergence of autism proteins at the cilium

doi: 10.1101/2024.12.05.626924

Figure Lengend Snippet: (A) Human GFP-tagged GRIN2B protein (neuronal communication annotation) localizes to cilia (axonemes labeled by acetylated alpha tubulin, magenta; basal bodies labeled by centrin-CFP, cyan) when expressed in X. laevis . (B) Human GFP-tagged constructs for control plasmids containing empty vector with GFP tagged at the N or C terminus do not (green) localize to primary cilia (Flag-tagged Dopamine D1 receptor, magenta; basal bodies labeled by FGFR1OP, red) when expressed in primary rat striatal neurons. (C) Endogenous antibody staining for autism associated neuronal communication proteins STXBP1 and GRIN2B in primary cilia (labeled by ARL13B, magenta; basal bodies labeled by FGFR1OP, red) in primary rat striatal neurons.

Article Snippet: Primary antibodies used: acetylated alpha-Tubulin (Sigma T6793, 1:600 for Xenopus , 1:1000 for rat brain slices), ARL13B (BiCell Scientific 90413h, 1:100 for human iPSC-derived neurons), CHD8 (Abcam ab114126, 1:100 for Xenopus and for rat striatal neurons), CHD2 (Novus Biologicals NBP2-32563, 1:100 for Xenopus and for rat striatal neurons), POGZ (Abcam ab167408, 1:100 for Xenopus and for rat striatal neurons), SYNGAP1 (MyBioSource MBS3216405, 1:100 for Xenopus ), SYNGAP1 (Abcam ab3344, 1:1000 for rat ependymal cells or human iPSC-derived neurons), SYNGAP1 (Invitrogen PA1-046, 1:500 for rat striatal neurons), STXBP1 (Synaptic Systems 116003, 1:500 for rat striatal neurons, 1:150 for rat ependymal cells), SHANK3 (Santa Cruz Biotechnology, sc-377088, 1:1000 for rat ependymal cells), PSD95 (Santa Cruz Biotechnology, sc-32290, 1:200 for rat striatal neurons, 10% donkey serum block), SCN2A (Alomone labs ASC-002, 1:100 for rat striatal neurons), SLC6A1 (Novus Biologicals NBP1-89802, 1:100 for rat striatal neurons), GRIN2B (Abcam ab65783, 1:200 for rat striatal neurons), FGFR1OP (Abnova H00011116-M01, 1:500), FGFR1OP (ProteinTech 11343-1-AP, 1:100), ARL13B-647 (ProteinTech CL647-17711, 1:500), and anti-GFP (Novus NB100-1614, 1:1000).

Techniques: Labeling, Construct, Control, Plasmid Preparation, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: FOP Negatively Regulates Ciliogenesis and Promotes Cell Cycle Re-entry by Facilitating Primary Cilia Disassembly

doi: 10.3389/fcell.2020.590449

Figure Lengend Snippet: FOP negatively regulates primary cilia growth. (A) The protein levels of FOP decrease during cilia assembly. RPE1 cells were serum-starved to induce primary cilia formation. The levels of FOP during cilia assembly were determined by immunoblotting at 0, 24, and 48 h. (B) RPE1 cells were transfected with negative control siRNA (siNC) or FOP siRNAs (siFOP). The knockdown efficacy was determined by immunoblotting at 72 h post-transfection. (C) Schematic diagram of the experimental design for the cilia assembly assay. (D) RPE1 cells transfected with siNC or FOP siRNAs were serum-starved and immunostained for FOP (red) and acetylated α-tubulin (Ac-Tub; green) or Arl13b (red) and γ-tubulin (γ-Tub; green). The nuclei were stained with DAPI. Scale bars, left, 20 μm; right, 10 μm. (E) Quantification of FOP intensity at the ciliary base described in (D) ; n = 105, 104 for siNC or siFOP treated cells, respectively; **** p < 0.0001 (unpaired, two-tailed Student t -test). (F) Quantification of the percentage of ciliated cells described in (D) . At least 200 cells per sample were analyzed in each experiment. (G) Quantification of the ciliary length in negative control cells (siNC) and FOP knockdown cells (siFOP); n = 327 and 325 for the negative control cells and FOP-depleted cells, respectively; ns, not significant; **** p < 0.0001 (unpaired, two-tailed, Student’s t -test). (H) RPE1 cells expressing GFP or FOP-GFP were transfected with the indicated siRNAs. The knockdown efficacy was determined by immunoblotting at 72 h post-transfection. (I) RPE1 cells expressing GFP or FOP-GFP were transfected with the indicated siRNAs, followed by 48 h serum starvation and immunostaining for γ-tubulin (γ-Tub; green) and Arl13b (red). The nuclei were stained with DAPI. Scale bar, 10 μm. (J) Quantification of the ciliary length in cells as described in (J) ; from left to right, the cilia number n = 125, 119, 148, 131, 137, and 123; **** p < 0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). (K) Serum-starved RPE1 cells transfected with siNC or FOP siRNAs were immunostained for FOP (red) and acetylated α-tubulin (Ac-Tub; green). The nuclei were stained with DAPI. Scar bars, 2 μm. (L) The inverse relationship between the FOP intensity and ciliary length. Each red dot represents a single measurement from an individual cell ( n = 55 cells). The green dotted line shows a linear fit through the data. Spearman correlation co-efficient (rs) and p -value are shown. Data are presented as mean ± SD from three independent experiments.

Article Snippet: The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).

Techniques: Western Blot, Transfection, Negative Control, Staining, Two Tailed Test, Expressing, Immunostaining

Journal: Frontiers in Cell and Developmental Biology

Article Title: FOP Negatively Regulates Ciliogenesis and Promotes Cell Cycle Re-entry by Facilitating Primary Cilia Disassembly

doi: 10.3389/fcell.2020.590449

Figure Lengend Snippet: Inhibition of AURKA rescues FOP-induced ciliogenesis defects. (A) Serum-starved vector control cells (GFP) and FOP-overexpressed cells (FOP-GFP) were immunostained for GFP (green) and Arl13b (red). The nuclei were stained with DAPI. Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells. At least 200 cells per sample were analyzed in each experiment. (C) Quantification of the ciliary length in vector control cells and FOP-overexpressed cells; n = 325 and 282 for vector control cells and FOP-overexpressed cells, respectively; ns, not significant; ** p < 0.01; **** p < 0.0001 (unpaired, two-tailed, Student’s t -test). (D) RPE1 cells expressing GFP or FOP-GFP were treated with DMSO or 1 μM AURKA inhibitor, PHA680632, serum-starved, and immunostained for acetylated α-tubulin (Ac-Tub; red) and γ-tubulin (γ-Tub; green). The nuclei were stained with DAPI. Scale bar, 5 μm. (E) Quantification of the percentage of ciliated cells described in (D) . At least 200 cells per sample were analyzed in each experiment; ns, not significant; ** p < 0.01, *** p < 0.001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). (F) Quantification of the ciliary length described in (D) . From left to right, the cilia number n = 170, 134, 146, and 144; ns, not significant; **** p < 0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). (G) RPE1 cells were transfected with siNC or AURKA siRNAs (siAURKA). The knockdown efficacy was determined by immunoblotting at 72 h post-transfection. (H) RPE1 cells expressing GFP or FOP-GFP were transfected with siNC or AURKA siRNAs, serum-starved, and immunostained for Arl13b (red) and GFP (green). The nuclei were stained with DAPI. Scale bar, 5 μm. (I) Quantification of the percentage of ciliated cells described in (H) . At least 200 cells per sample were analyzed in each experiment; ns, not significant; **** p < 0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). (J) Quantification of the ciliary length described in (H) . From left to right, the cilia number n = 197, 174, 173, and 165; ns, not significant; **** p < 0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). (K) AURKA levels in serum-starved GFP- and FOP-GFP-expressed cells. (L) Quantification of relative AURKA levels described in (J) ; ** p < 0.01 (unpaired, two-tailed, Student’s t -test). (M,N) qRT-PCR analysis of FOP (M) and AURKA (N) mRNA levels in GFP- and FOP-GFP-expressing cells; *** p < 0.001 (unpaired, two-tailed, Student’s t -test). (O) RPE1 cells stably expressing GFP or FOP-GFP were serum-starved and immunostained for AURKA (red) and acetylated α-tubulin (Ac-Tub; green). The nuclei were stained with DAPI. Scale bars, 5 μm (panel) and 2 μm (insert). (P) Quantification of the percentage of AURKA positive cells described in (O) . At least 100 cells per sample were analyzed in each experiment; ** p < 0.01 (unpaired, two-tailed, Student’s t -test). Data are presented as mean ± SD from three independent experiments.

Article Snippet: The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).

Techniques: Inhibition, Plasmid Preparation, Staining, Two Tailed Test, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Stable Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: FOP Negatively Regulates Ciliogenesis and Promotes Cell Cycle Re-entry by Facilitating Primary Cilia Disassembly

doi: 10.3389/fcell.2020.590449

Figure Lengend Snippet: FOP-promoted cilia disassembly during cell cycle re-entry is dependent on AURKA. (A) Immunoblotting analysis of the total levels of AURKA in siNC or siFOP treated cells at 0, 6, 12, 18, and 24 h post serum re-stimulation. (B) Quantification data described in (A) ; ns, not significant; ∗∗ p < 0.01; **** p < 0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). (C,D) qRT-PCR analysis of FOP (C) and AURKA (D) mRNA levels in siNC and siFOP-treated cells; ∗∗∗ p < 0.001, **** p < 0.001 (unpaired, two-tailed, Student’s t -test). (E) RPE1 cells transfected with siNC or siFOP were immunostained for AURKA (red) and acetylated α-tubulin (Ac-Tub; green). The nuclei were stained with DAPI. Scale bar, 5 μm. (F) Quantification of AURKA intensity at the ciliary base described in (E) ; n = 45, 44 for siNC or siFOP treated cells, respectively; **** p < 0.0001 (unpaired, two-tailed Student t -test). (G) RPE1 cells expressing GFP or FOP-GFP were transfected with siNC or AURKA siRNAs and subjected to cilia disassembly assay. Cells were fixed at the indicated time points and immunostained for Arl13b and γ-tubulin. The percentage of ciliated cells were quantified. At least 100 cells per sample were analyzed in each experiment; ns, not significant; * p < 0.05; ∗∗ p < 0.01; **** p < 0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons test). Data are presented as mean ± SD from three independent experiments.

Article Snippet: The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).

Techniques: Western Blot, Quantitative RT-PCR, Two Tailed Test, Transfection, Staining, Expressing