Journal: Science Advances

Article Title: Inhibition of focal adhesion kinase impairs tumor formation and preserves hearing in a murine model of NF2-related schwannomatosis

doi: 10.1126/sciadv.ady8382

Figure Lengend Snippet: ( A ) Scatter plot depicting DRG volumes (in cubic millimeters) from 10-month-old mice of the indicated genotypes ( WT , n = 14; Fak KO , n = 12; Nf2 , n = 12; Nf2Fak +/− , n = 16; DKO n = 18). Dots represent individual DRG ( n = 4 per mouse). Error bars reflect the SEM. P values represent one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. ( B ) Bar plot depicting the average ABR threshold measured in decibels of mice with the indicated genotypes at 4 and 10 months of age ( WT , n = 11; Fak KO , n = 13; Nf2 , n = 11; Nf2Fak +/− , n = 11; DKO n = 13). P values represent one-way ANOVA with Šídák’s multiple-comparison test. Error bars reflect the SEM. ( C ) Bar plot depicting a ~2-fold RR of hearing impairment (average ABR threshold ≥ 55 dB) at 10 months of age among Nf2 and DKO mice. RR = 1.957, 95% CI (1.301 to 2.992), and P = 0.0016. P value reflects two-sided, Fisher’s exact test. ( D ) Representative photomicrographs of hematoxylin and eosin (H&E)–stained DRG obtained from 10-month-old Nf2 and DKO mice. Mask in the bottom panel and depicts abnormal tissue (red), neuron cell bodies (blue), and nerve (lavender). ( E and F ) Plots comparing the percentage of abnormal tissue (E) and neuron cell bodies (F) in Nf2 and DKO DRGs. Dots represent individual DRG. Error bars reflect the SEM. P values represent unpaired, two-tailed t tests between groups. ( G ) Representative photomicrographs of H&E-stained tissue sections comparing the architecture of Nf2 and DKO DRGs. ( H ) Representative photomicrographs of Masson’s trichrome–stained DRG obtained from Nf2 and DKO mice. Mask in the bottom panel and depicts collagen staining in purple. ( I ) Plot comparing collagen within the 10-month-old Nf2 and DKO DRGs, reflected as percentage of the total tissue area analyzed. Dots represent individual DRG. Error bars reflect the SEM. P values represent unpaired, two-tailed t test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: The 02.3 cell line was transfected with equal amounts of a CRISPR-Cas9 KO plasmid pool targeting NF2 and a homology-directed repair (HDR) plasmid pool purchased from Santa Cruz Biotechnology (sc-400504 and sc-400504-HDR, respectively).

Techniques: Comparison, Staining, Two Tailed Test

Journal: Science Advances

Article Title: Inhibition of focal adhesion kinase impairs tumor formation and preserves hearing in a murine model of NF2-related schwannomatosis

doi: 10.1126/sciadv.ady8382

Figure Lengend Snippet: ( A to C ) Gene set enrichment plots depicting negative enrichment of the Hallmark inflammatory response (A), IL-6–JAK–STAT3 (B), and interferon-γ signaling (C) gene signatures by DKO DRGs. Black vertical bars indicate the rank of genes comprising the gene signature. Black curve corresponds to the running enrichment score for the gene set. Normalized enrichment score (NES) and q value for DKO compared to Nf2 DRG are as shown. ( D ) Cell type mapping panel representing inferred cell types in Nf2 and DKO DRG. ( E ) Representative photomicrographs of DRG immunohistochemically stained for F4/80. HALO cytonuclear mask is shown in the bottom panel. Blue cells indicate negative staining for F4/80, yellow cells indicate weak positive (1+), orange cells indicate moderate positive (2+), and red cells indicate strong positive (3+). ( F ) Bar plot depicting the percentage (%) of cells exhibiting positive staining of F4/80. Dots indicate individual DRG. Error bars reflect the SEM. P value represents unpaired, two-tailed t test. ( G to J ) Scatter plots comparing log 2 [counts per million (CPM) + 4] normalized counts of Lgals2 (G), Ccr5 (H), Cxcl3 (I), and Nlrp3 (J) in Nf2 and DKO DRG. Dots represent individual mice. Error bars reflect the SEM. P values represent unpaired, two-tailed t test. ( K ) Uniform manifold approximation and projection (UMAP) visualization depicting the expression of NLRP3 across distinct cell populations in human VS. Color overlays indicate NLRP3 transcript levels per cell. Gradient of gray to indigo indicates relative abundance of transcripts per cell, with indigo indicating higher expression and gray signifying a lack of detection. ( L ) Representative images of IF staining of F4/80 (red) and NLRP3 (green) in a murine DRG. 4′,6-diamidino-2-phenylindole (DAPI) is blue. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Macrophages (Mac1 and Mac2) and dendritic cells (DC1 and DC2).

Article Snippet: The 02.3 cell line was transfected with equal amounts of a CRISPR-Cas9 KO plasmid pool targeting NF2 and a homology-directed repair (HDR) plasmid pool purchased from Santa Cruz Biotechnology (sc-400504 and sc-400504-HDR, respectively).

Techniques: Staining, Negative Staining, Two Tailed Test, Expressing

Journal: Science Advances

Article Title: Inhibition of focal adhesion kinase impairs tumor formation and preserves hearing in a murine model of NF2-related schwannomatosis

doi: 10.1126/sciadv.ady8382

Figure Lengend Snippet: ( A ) Bar plot of Nlrp3 transcript expression [reads per kilobase of transcript per million mapped reads (RPKM)] at indicated days of embryonic [embryonic day 13.5 (E13.5) and E17.5] and postnatal [postnatal day 1 (P1), P5, P14, P24, and P60] development. ( B to D ) UMAPs depicting the expression of Nlrp3 across distinct cell populations in the murine sciatic nerve 1 (C) and 3 (D) days postinjury. Nlrp3 expression in the naïve murine sciatic nerve serves as a reference (B). ( E to H ) Gene set enrichment plots depicting negative enrichment of the indicated gene signatures by DKO DRG. Black vertical bars indicate the rank of genes comprising the gene signature. Black curve corresponds to the running enrichment score for the gene set. NES and q value reflect DKO compared to Nf2 DRG. GO, Gene Ontology. ( I and J ) UMAPs depicting the expression of TNF (I) and IL-1B (J) across distinct cell populations in human VS. ( K ) Bar plot depicting Hallmark pathway enrichment analysis of the top 20 biological pathways enriched in NLRP3 + compared to NLRP3 – macrophages in human VS. X-axis represents the NES, with positive values indicating up-regulation and negative values indicating down-regulation. Pathways are ranked by significance. Turquoise bars represent pathways that meet the significance threshold (adjusted P < 0.05). adj., adjusted. ( L ) Representative photomicrographs of DRG immunohistochemically stained for IL-6. The HALO cytonuclear mask is shown in the bottom panel. Blue cells indicate negative staining for IL-6, yellow cells indicate weak positive (1+), orange cells indicate moderate positive (2+), and red cells indicate strong positive (3+). ( M ) Bar plot depicting the percentage of cells exhibiting strong and moderate staining of IL-6. Dots indicate individual DRG. Error bars reflect the SEM. P value represents unpaired, two-tailed t test. Color overlays indicate transcript levels per cell. Gradient of gray to indigo indicates relative abundance of transcripts per cell, with indigo indicating higher expression and gray signifying a lack of detection. mod, moderate. * P ≤ 0.05.

Article Snippet: The 02.3 cell line was transfected with equal amounts of a CRISPR-Cas9 KO plasmid pool targeting NF2 and a homology-directed repair (HDR) plasmid pool purchased from Santa Cruz Biotechnology (sc-400504 and sc-400504-HDR, respectively).

Techniques: Expressing, Staining, Negative Staining, Two Tailed Test

Journal: Science Advances

Article Title: Inhibition of focal adhesion kinase impairs tumor formation and preserves hearing in a murine model of NF2-related schwannomatosis

doi: 10.1126/sciadv.ady8382

Figure Lengend Snippet: ( A and B ) UMAPs depicting the expression of MET (A) and HGF (B) in human VS. Color overlays indicate MET or HGF transcript levels per cell. Gradient of gray to indigo indicates relative abundance of transcripts per cell, with indigo indicating higher expression and gray signifying a lack of detection. ( C and D ) Bar plot depicting the weighted contributions of MET (C) and HGF (D) expression across distinct cell populations in human VS. ( E and F ) GSEA plots depicting positive enrichment of the indicated pathways by MET + SCs in human VS. NES and adjusted P values are shown. ( G ) Circle plot depicting the HGF signaling network. Connecting ribbons reflect predicted signaling interactions, with ribbon width proportional to interaction strength and arrows indicating directionality. ( H ) Heatmap depicting the communication probability scores between cell pairs within the HGF signaling network. Gradient indicates the strength of predicted cell-cell communication. Prob., probability. ( I ) Matrix depicting cellular functional classification in the HGF signaling network. Color intensity reflects the importance score for each role, with 1 signifying the highest importance and 0 the lowest. ( J and K ) Scatter plots comparing expression of Met (J) and Spint1 (K) in Nf2 and DKO DRG. Dots represent individual mice. Error bars reflect the SEM. P values represent unpaired, two-tailed t test. ( L ) Photomicrographs of DRG stained for MET. HALO mask is shown in the bottom panel. Yellow indicates weak positive (1+), orange indicates moderate positive (2+), and red indicates strong positive (3+) staining for MET. ( M ) Box and whisker plot depicting MET protein expression in DRG obtained from Nf2 and DKO mice. Dots represent individual DRG. Error bars represent the 95% CI. P value represents unpaired, two-tailed t test. Nonmyelinating SC (nmSC), myelinating SC (mSC), fibroblasts (Fb), macrophages (Mac1 and Mac2), endothelial (Endo), natural killer/T (NK/T), dendritic (DC1, DC2), B (B), and perivascular/vascular smooth muscle (Peri/VS) cells. ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: The 02.3 cell line was transfected with equal amounts of a CRISPR-Cas9 KO plasmid pool targeting NF2 and a homology-directed repair (HDR) plasmid pool purchased from Santa Cruz Biotechnology (sc-400504 and sc-400504-HDR, respectively).

Techniques: Expressing, Functional Assay, Two Tailed Test, Staining, Whisker Assay

Journal: Science Advances

Article Title: Inhibition of focal adhesion kinase impairs tumor formation and preserves hearing in a murine model of NF2-related schwannomatosis

doi: 10.1126/sciadv.ady8382

Figure Lengend Snippet: ( A and B ) Dose-response curves comparing the effects of defactinib and a FAK-targeting PROTAC on the viability in human (A) and murine (B) NF2-deficient SCs. Cells were treated with increasing concentrations (0 to 20 μM) of either defactinib or PROTAC for 48 hours, at which time viability was determined via CellTiter-Glo assay, and luminescent values were normalized to untreated control (100%). The dotted line indicates 50% viability. Data points represent mean values ± SEM of n = 12 (02.3 NF2 −/− KO3) or n = 4 (MSO3) replicates. ( C ) Colony formation assays stained with methylene blue and showing the effect of increasing concentrations of defactinib (1 to 5 μM) compared to dimethyl sulfoxide (DMSO) control on three NF2-deficient SC lines: 02.3 NF2 −/− K03, HEI-193, and MS03. ( D ) Volcano plot showing changes in MIB binding of kinases following 24-hour treatment with 1 μM defactinib compared to DMSO control in MS03 cells. X axis represents log 2 fold change (FC) in binding, and the y axis shows the negative log 10 of the P value. Dotted lines indicate significance thresholds for FC and P value. ( E ) Scatter plot depicting DRG volumes (in cubic millimeters) from mice treated with VS-4718 ( n = 14) or vehicle control ( n = 11). Dots represent individual DRG ( n = 4 per mouse). P value represents unpaired, two-tailed t test. Error bars reflect the SEM. ( F ) Representative photomicrographs of H&E-stained DRG obtained from Nf2 and DKO mice. HALO AI DenseNet mask is shown in the bottom panel and depicts abnormal tissue (red), neuron cell bodies (blue), and nerve (lavender). ( G and H ) Plots comparing the percentage of abnormal tissue (G) and neuron cell bodies (H) in Nf2 and DKO DRG. Dots represent individual DRG. Error bars reflect the SEM. P values represent unpaired, two-tailed t test. *** P ≤ 0.001 and n.s., not significant.

Article Snippet: The 02.3 cell line was transfected with equal amounts of a CRISPR-Cas9 KO plasmid pool targeting NF2 and a homology-directed repair (HDR) plasmid pool purchased from Santa Cruz Biotechnology (sc-400504 and sc-400504-HDR, respectively).

Techniques: Glo Assay, Control, Staining, Binding Assay, Two Tailed Test

Journal: Science Advances

Article Title: Inhibition of focal adhesion kinase impairs tumor formation and preserves hearing in a murine model of NF2-related schwannomatosis

doi: 10.1126/sciadv.ady8382

Figure Lengend Snippet: ( A ) Western blot depicting pERK, total ERK, and phospho-FAK (pFAK) Tyr 397 expression in nerves obtained from mice treated with VS-4718 ( n = 3) or vehicle control ( n = 3) for 2 weeks. Vinculin serves as a loading control. ( B to D ) Bar graphs quantifying pFAK Tyr 397 (B), pERK (C), and total ERK (D) expression normalized to vinculin from the blots depicted in (A). Dots represent individual mice. Error bars reflect SEM. P value represents unpaired, two-tailed t test. tERK, total ERK. ( E and F ) Western blots depicting the expression of pFAK Tyr 397 , total FAK, cleaved caspase-3, and cleaved PARP (E), as well as the proliferation markers Ki-67 and PCNA (F) in a murine Nf2 -deficient SC line (MS03) treated with 1 μM VS-4718 and selumetinib alone or in combination. DMSO serves as the control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 are loading controls. ( G to I ) Heatmaps depicting Zero Interaction Potency (ZIP) (G), Loewe (H), and Bliss (I) synergy scores for combination defactinib plus selumetinib in murine NF2-deficient SCs (MS03). Viability (%) was evaluated at 48 hours by CellTiter-Glo and normalized to the untreated control. Data represent the mean of three replicates for each condition. Color scale indicates synergy scores from −30 (blue; antagonistic) to +30 (yellow; synergistic). Heatmaps and corresponding mean synergy scores were calculated with their respective statistical significance (ZIP: 13.82, P = 4.45 × 10 –13 ; Loewe: 14.16, P = 1.18 × 10 –12 ; Bliss: 14.14, P = 2.69 × 10 –13 ) were computed using SynergyFinder+. ( J and K ) Colony formation assay stained with methylene blue (J) and dot plot (K) showing the effect of combination defactinib (1 μM) plus selumetinib (1 μM) compared to the single agents and DMSO control in a murine Nf2 -deficient SC line (MS03). Dots represent independent samples. Error bars represent the SEM. P value represents unpaired, two-tailed t tests between groups. * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001, and n.s., not significant.

Article Snippet: The 02.3 cell line was transfected with equal amounts of a CRISPR-Cas9 KO plasmid pool targeting NF2 and a homology-directed repair (HDR) plasmid pool purchased from Santa Cruz Biotechnology (sc-400504 and sc-400504-HDR, respectively).

Techniques: Western Blot, Expressing, Control, Two Tailed Test, Colony Assay, Staining

Journal: Oncogene

Article Title: KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals.

doi: 10.1038/onc.2012.196

Figure Lengend Snippet: Figure 4. KIBRA expression antagonises YAP-induced transcriptional regulation. (a) mRNA expression of the YAP target genes BMP2, FGF1, RASSF4 and PDGFb was assessed by quantitative real-time PCR in MCF10A-YAP cells overexpressing KIBRA, Willin or Merlin. mRNA levels were compared with the empty vector control MCF10A-YAP cells. Willin and KIBRA overexpression increased BMP2 and RASSF4 mRNA levels and decreased FGF1 and PDGFb mRNA levels. Means were calculated from Ct values from two analyses conducted in triplicate. Error bars represent ±s.e. (n ¼ 6). MCF10A-YAP-vector vs MCF10A-YAP-KIBRA or MCF10A-YAP-Willin for all the analysed genes: *Po0.05, **Po0.01, ***Po0.001 Student’s t-test. MCF10A-YAP-vector vs MCF10A-YAP-Merlin: CTGF (P ¼ 0.129), FGF1 (P ¼ 0.005), BMP2 (P ¼ 0.585), RASSF4 (P ¼ 0.307), PDGFb (P ¼ 0.06), Student’s t-test. (b) Immunoblot analysis of KIBRA, Willin or Merlin overexpression in MCF10A-YAP cells, showing 2.7, 1.8 and 8.6-fold increases compared to empty vector control. b-actin was used as a loading control.

Article Snippet: All experiments were carried out at 80–90% confluency. pBABE Nf2 (Johnson et al.45) was obtained from Addgene (plasmid 14116).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Over Expression, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: Clinical and molecular features of patients with tumor molecular profiling on biopsies obtained upon resistance to ALK inhibitors in the MATCH-R study.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Biomarker Discovery, Sequencing, RNA Sequencing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: NF2 loss of function mediates resistance to lorlatinib. A, Clinical course of patient MR135 and mutational profile of samples obtained on lorlatinib progression (PD, progressive Disease). B, Cell survival assay assessed with Cell Titer Glo of MR135 lorlatinib resistant cells from biopsy 1 (MR135-R1) treated for 7 days with the indicated concentrations of lorlatinib and vistusertib (AZD2014) alone or in combination. C, Immunoblot analysis from cell lysates of MR135-R1 treated for 24hs with the specified doses of lorlatinib, vistusertib (AZD2014) and ponatinib alone or in combination using indicated antibodies. D, Athymic nude mice bearing MR135-R2 PDX were administered lorlatinib or vistusertib 20 mg/kg orally. Tumor volumes, mean ±SD (n =8); (*** p < 0.001). E, Cell lysates from H3122 parental and H3122 cells with NF2 heterozygous deletions or homozygous deletions, generated by CRISPR-CAS9 gene editing, were immunoblotted to detect merlin expression. H3122 cells with bi-allelic NF2 knock-out lacked merlin expression. F, Cell survival assay of H3122 parental and H3122 NF2 knock-out (NF2 KO) cells treated with lorlatinib for 7 days. Cell survival was assessed by Cell Titer Glo. G, Cell proliferation assay of H3122 parental and H3122 NF2 KO cells untreated and treated with lorlatinib measured at baseline, day 2, day 5 and day 7. Cell viability was assessed with Cell Titer Glo. H, Caspase 3/7 activation (Caspase 3/7-Glo assay) relative to the number of live cells simultaneously assessed in the cell proliferation assay previously described. I, H3122 parental and NF2 KO cells were treated with the indicated doses of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Clonogenic Cell Survival Assay, Western Blot, Generated, CRISPR, Expressing, Knock-Out, Proliferation Assay, Activation Assay, Glo Assay