EMBO/soda-nel huggingface dataset:
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 3

Figure 3. Ser259-MYRF phosphorylation is increased in PLP-150Q mice and HD patient brains

Anti-Ser259-MYRF antibody was generated using the peptides containing pS259.

Western blotting analysis of the corpus callosum in PLP-23Q or PLP-150Q mice with anti-pS259 and an antibody to total MYRF. Note that LAQ (5 mg/kg) treatment for 2 months decreased the phosphorylation in both full length (fMYRF) and N-terminal MYRF (nMYRF) in PLP-150Q mice.

Ratios of phosphor-S259-MYRF (pMYRF) and total MYRF to GAPDH. The ratios were obtained from 3 independent western blotting experiments. Student's t-test; ***P=0.0007. Data are mean ±SEM.

Anti-Ser259-MYRF immunohistochemical staining of the corpus callosum (CC, indicated between two dotted lines) of PLP-23Q and PLP-150Q mice showing decreased expression of Ser259-MYRF by LAQ (5 mg/kg) treatment for 2 months. Micrographs at 10X (upper) and 40X (lower) are shown. Scale bars: 40 μm.

Quantitative statistics of the cells stained by anti-Ser259 MYRF in each filed was presented. Twenty random fields (40X) of each section were examined to count anti-Ser259 positive cells, n=3 mice in each group. One-way ANNOVA with Tukey's test. ***P=0.0003; **P=0.0039. Data are mean ±SEM.

Information of postmortem brains of control and HD patients.

Western blotting analysis of MBP and Ser259-MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non-HD control individuals. Ratios of MBP to GAPDH, or Ser259 (pMYRF) to total MYRF are presented on the right. One-way ANNOVA with Tukey's test. MBP: ***P=0.0004; pMYRF: **P=0.0051. Data are mean ±SEM.

Western blotting analysis of the expression of Ser259 (pMYRF) and total MYRF (full-length: fMYRF and N-terminal: nMYRF) in 13-months-old HD-KI and WT mouse cortex. Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots. One-way ANNOVA with Tukey's test. ***P=0.00083. Data are mean ±SEM.
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 4

Figure 4. PRKG2 is the potential candidate kinase for phosphorylation of S259 in MYRF

Heatmaps of gene expression in PLP-150Q mouse corpus callosum at 5 months after LAQ (5 mg/kg) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes.

Most significant gene networks of different functions via GO-cluster and categorization. The bold-type indicated two phosphorylation associated clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS".

The individual networks of up-regulated gene were heat mapped typically, on the clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC _PROCESS".

Identification of consensus sequences from PRKG2 kinase substrate peaks, in which the same motif was identified using the 15 amino acid sequences surrounding the strongest 60 peaks. The PRKG2 substrate core consensus sequences of (R/K)2-3-X-S/T-X, where S=serine, R=arginine, K=lysine, T=threonine, X standing for any random amino acid, are mostly consistent with MYRF amino acid 252-266. The rat, monkey and human MYRF have the highly conserved core sequences of "KKRKHSE".

Western blotting showing down-regulation of PRKG2 in PLP-150Q mouse corpus callosum at 3 months of age by 5 mg/kg LAQ. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. PRKG2: **P=0.0076(left) and ***P=0.0015(right); p-VASP: ***P=3.86x10-5(left) and 1.01x10-5(right). Data are mean ±SEM.

Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 mg/kg) treatment for 2 months. Only PRKG2 was decreased by LAQ. n=3 mice in each group. Student's t-test; **P=0.0073; ***P=0.00041. Data are mean ±SEM.
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 5

Figure 5. LAQ suppresses PRKG2 gene transcript expression via AhR negative regulation

The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (-472/+131) was determined for transcription activity in cultured N2a cells, obtained from 3 independent experiments. One-way ANNOVA with Tukey's test; ***P<0.001. Data are mean ±SEM.

The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P<0.001. Data are mean ±SEM.

The putative cis-elements (in box and sequences below) in the PRKG2 promoter were determined by TFSEARCH, ConTra and ALGGEN program analyses. Six AhR transcriptional factor binding sites were predicted according to the conserved sequence of "CACGC" or "GCGTG".

The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n=3 mice in each group. Student's t-test; Cyp1a1: ***P=0.00042; Ugt1a6a; ***P=0.00057. Data are mean ±SEM.

The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.

The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The results were obtained from three independent experiments. One-way ANNOVA with Tukey's test. LAQ: ***P=0.00065; 2,3,7,8-TCDD: ***P=0.00014. Data are mean ±SEM
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 6

Figure 6. The activation or over-expressing of PRKG2 promoted the phosphorylation of Ser259-MYRF

Western blotting showing that the phosphorylation of transfected N-terminal MYRF (pMYRF) was promoted by treatment with the PKG activator of 8-Br-cGMP (10 or 100 μM) for 1 hr or co-transfected 150Q-HTT for 24 hr, which was blocked by treatment of the PKG inhibitors of RKRARKE (50 μM) or KT5823 (5 μM) for 12 hr. trans-nMYRF: transfected N-terminal MYRF.

Adeno-associated virus vector expressing full-length PRKG2 under the control of the CMV promoter.

Western blotting of N2a cell line that was transfected with AAV-PRKG2 for 48 hr. The blots were probed with anti-Ser259 (pMYRF) antibody. Note that overexpression of PRKG2 increased phosphorylation of N-terminal MYRF (nMYRF) in a dose-dependent manner.

Stereotaxic injection of AAV-GFP virus into the corpus callosum (CC, indicated between two dotted lines) in the mouse brain. Ctx: cortex, Str: striatum. Scale bar: 100 μm..

Immunohistochemical staining with anti-Ser259 (pMYRF) showing that AAV-PRKG2, but not AAV-GFP, promotes MYRF phosphorylation in the corpus callosum. Scale bar: 40 μm.

Quantitative analysis of the pMYRF- or total MYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=8.79x10-5. Data are mean ±SEM.

Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV-PRKG2, but not AAV-GFP (green), promotes MYRF phosphorylation (red) in the injected corpus callosum. Scale bar: 40 μm.

Western blotting analysis of the mouse corpus callosum tissues using anti-Ser259 (pMYRF), phosphor-PRKG2 (pPRKG2) or phosphor-VASP (pVASP). Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 7

Figure 7. Knocking down PRKG2 alleviated demyelination and decreased phosphorylation of MYRF in PLP-150Q/Cas9 mice

Construction of AAV-U6-gRNA-CMV-RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV-gRNA-PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP-150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 μm

Western blotting analysis of the PLP-150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor-PRKG2 or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots. Student's t-test; pMYRF: ***P=0.00026; MBP; **P=0.0036. Data are presented as mean ±SEM. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.

Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP-150Q mouse. Scale bar: 40 μm.

Quantitative analysis of the pMYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=0.00022. Data are mean ±SEM.

Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV- PRKG2-gRNA (red) injection increased MBP expression (green) as compared with AAV-gRNA-control. Scale bar: 40 μm.

Quantitative analysis of the MBP-positive cells in the corpus callosum injected with control-gRNA or PRKG2-gRNA. Twenty random fields (40X) in each section were examined. n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=4.43x10-5. Data are mean ±SEM.

A proposed model for the protective effect of LAQ in HD mice. LAQ increases myelin protein expression by reducing the expression of PRKG2, leading to reduced phosphorylation of MYRF and its dissociation from mutant HTT and therefore increasing its transcriptional activity to express myelin associated genes.

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Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 1

Figure 1 - Identification of HOXA4 as a negative regulator of YAP/TEAD transcriptional activity through a pooled shRNA screen.

A, Dynamic fluorescence patterns according to cell density in 8×GTIIC-EmGFP 293T cells.

B, EmGFP intensity in LATS2 knockdown, or forced expression of a constitutively active form of YAP (YAP-S127A). Scale bars indicate 100 μm.

C, Schematic drawing of FACS-based pooled shRNA screen of molecules that affect the YAP/TEAD signaling pathway in 8×GTIIC-EmGFP 293T cells.

D, Scatter plots of mean frequency of each shRNA per gene. Average of v HOXA4 is indicated as a red dot.

E, Augmented 8×GTIIC-luciferase reporter activity by loss of HOXA4. ShSAV1 was used as an experimental control. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.

F and G, Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4 (F) or HOXA4 expression vector (G). **P<0.01, by unpaired two-tailed Student's t-test.

Data information: Graphs of (E-F) show mean ± SEM; three biological repeats.
Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 2

Figure 2 - HOXA4 negatively regulates YAP/TEAD transcriptional activity independently of YAP phosphorylation.

A, Quantitative real-time PCR analysis of CTGF, CYR61, and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4. ShLATS1/2 and shYAP were used as an experimental control. *P<0.05, **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.

B, 8×GTIIC-luciferase reporter assays in HEK 293T cells transfected HOXA4 and/or shLATS1/2. **P<0.01 vs shCtrl, by two-way ANOVA with Sidak's correction.

C, Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with overexpression of HOXA4 and/or constitutive active form of YAP (YAP -5SA). *P<0.05, **P<0.01 vs YAP-5SA with LacZ, by ANOVA with Sidak's correction.

D, Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and/or overexpression of YAP-5SA. *P<0.05, **P<0.01 vs YAP-5SA with shCtrl, by ANOVA with Sidak's correction.

Data information: All data are presented as mean ± SEM; three biological repeats.
Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 3

Figure 3 - Protein-protein interactions between HoxA4 and TEADs.

A and B, Association of HOXA4 with TEAD1 (A) but not YAP (B) indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-TEAD1 or Myc-YAP.

C, Interaction between endogenous TEAD1 and HOXA4 by co-IP experiments in HEK 293T cells. The interaction between endogenous TEAD1 and YAP was used as a positive control.

D, Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1.

E, Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).

F, Requirement of TEAD1 (a.a. 30-135) for its interaction with HOXA4 via its homeodomain (a.a. 216-274).

G, Failure of TEAD1 (a.a. 30-102), TEA domain alone, to interact with HOXA4.

H and I, Diagram showing the structure of truncated HOXA4 (H) and TEAD1 (I) constructs and possible protein-protein interactions between them. Conservation between human TEAD1, 2, 3, and 4 is shown (I).

Data information: All experiments above were repeated at least twice.
Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 4

Figure 4 - HOXA4 negatively regulates TEAD-YAP interaction.

A, ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.

B, Competitive co-IP assay and densitometry showing significantly reduced TEAD-YAP interaction by overexpression of HOXA4 in a dose-dependent manner in HEK 293T cells. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.

C, Working hypothesis whereby HOXA4 inhibits YAP/TEAD-mediated transcription activity.
Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 5

Figure 5 - HOXA4 regulates the phenotypic switching of vascular smooth muscle cells.

A, Expression of Hoxa4 in various mouse tissues using qRT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control. WAT; white adipose tissue, BAT; brown adipose tissue, Skm; skeletal muscle.

B. Quantitative real-time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF-BB stimulation for 24 hours. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.

C, Quantitative real-time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.

D and E, Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP/TAZ (D) or HOXA4 (E). Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.

F, Quantitative real-time PCR analysis of YAP/TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.

G, Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.

H, BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.

I, Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction.

J, BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.

Data information: Graphs are presented as means ± SEM; three biological repeats, except for (H) and (J).
Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 6

Figure 6 - A negative effect of HOXA4 on VSMC phenotypic switching is mediated by YAP/TEAD.

A, Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction.

B, Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.

C, Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.

D, ChIP assays of CTGF, CYR61 and α-SMA promoters using anti-FLAG-tag, anti-Myc-tag, anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P<0.05, **P<0.01 vs FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.
Homeobox A4 Suppresses Vascular Remodeling by Repressing YAP/TEAD Transcriptional Activity (2020)
Figure 7

Figure 7 - Hoxa4 KO mice alleviate smooth muscle phenotypic modulation and abrogate neointima formation after carotid artery ligation injury.

A, Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).

B, Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.

C, Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation (n=6).

D, Quantitative real-time PCR analysis of Yap/Tead target genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).

E, Quantitative real-time PCR analysis of Yap/Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.

F, Representative western blotting analysis and densitometry of non-ligated (right; R) and ligated (left; L) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation (WT: n=6, Hoxa4 KO: n=7). *P<0.05, **P<0.01, by two-way ANOVA with Sidak's correction.

G to I, Representative images showing Elastica van Gieson staining (G), and vessel, neointima and media area (H), and neointima to media layer ratio (I) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation (WT: n=14, Hoxa4 KO: n=11).

Data information: Images and data were obtained at 250μm proximal to the ligation. Scale bars indicate 100 μm. *P<0.05, **P<0.01 vs WT, except in (C) vs baseline, by unpaired two-tailed Mann-Whitney test. Data are presented as mean ± SEM. N.D.; not detected.

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ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 1

Figure 1 - ALKAL stimulates ALK signaling and transcriptional responses in NB cells.

A. RNA-Seq based differential gene expression (DE) was measured in NB1 and IMR32 NB cell lines in response to ALKAL2 stimulation. See Table S1 for detailed results.

B. Volcano plot showing DE 1hr after NB1 (top) and IMR32 (bottom) cell treatment with ALKAL2. Dashed lines show DE thresholds. Up-/downregulated genes indicated in blue. Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red.

C. Venn diagram indicating the number of DE genes between different conditions as indicated. Outer circles (labels below diagram) indicate the number of DE genes after ALKAL2 addition for NB1 cells (34 genes) and IMR32 cells (13 genes). Inner circles (labels on top) correspond to the number of DE genes after addition of lorlatinib. Six genes that are DE in both cell lines and sensitive to lorlatinib are indicated.

D. Temporal dynamics of ALKAL2-induced transcription of ARC, EGR1-3, FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib, as indicated.

E. Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells. Cells were treated for 0, 1 and 6h in the presence and absence of lorlatinib as indicated.

F. Transcription factor prediction based on a gene set enrichment analysis (GSEA) of the identified 6-gene set. Bar plot shows the log10(q) values of all enriched transcription factors at 10% FDR.

G. Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells. Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated. RNA-Seq analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.