EMBO/soda-nel huggingface dataset:
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 3

Figure 3 -CRMP4 protein levels are altered via a dynein-dependent activity

(A) Representative images of healthy or C9orf72 iPSC-derived MNs treated with Sema3A or untreated in the distal compartment, 6 hours post treatment. Red: denotes Tubulin, Green: denotes CRMP4. Scale bar: 5 μm.

(B) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC-derived MNs with Sema3A treatment or untreated. 14 untreated healthy axons, 12 healthy axons with Sema3A treatment, 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p = 0.0338; ****p<0.0001.

(C) Illustration of the experimental procedure for MNs in an MFC treated with the fluorescently tagged retrograde tracer CTX in the distal compartment. Neuronal cell bodies in the primary neuron whose axons have traversed into the distal compartment were also labeled by the retrograde tracer.

(D) Representative images of healthy or C9orf72 human-derived MN cell somata with Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Gray: denotes CTX, Green: denotes CRMP4. Scale bar: 5μm.

(E) Quantification of CRMP4 intensity (normalized to GAPDH+mCherry/area) levels at the somata of healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Analysis performed in 3 independent chambers per condition. 19 healthy untreated cell somata, 26 healthy cell somata with Sema3A treatment, 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n=3, Data presented as mean ±SE, *p=0.0207; ***p=0.0004.

(F) Representative images of healthy or C9orf72 human-derived MN proximal axons with Sema3A treatment, Sema3A + dynein inhibitor treatment or untreated control. Green: denotes CRMP4, Red: denotes GAPDH. Scale bar: 5μm.

(G) Quantification of CRMP4 intensity levels (normalized to GAPDH+mCherry/area) at the proximal axons in healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated control. Analysis performed from 3 independent chambers in each condition. 21 healthy untreated proximal axons, 24 healthy proximal axons with Sema3A treatment, 16 healthy proximal axons with Sema3A + dynein inhibitor treatment, 12 C9orf72 untreated proximal axons, 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n = 3, Data presented as mean ±SE, *p =0.0334, ****p<0.0001.
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 4

Figure 4 -CRMP4 binds dynein via a specific 50 amino acid motif

(A) Crystal structure (PDB code 4CNT) of a CRMP4 monomer (upper panel) and biological tetramer assembly (lower panel). The peptides that were selected to inhibit binding are highlighted and color coded as indicated.

(B) Upper panel represent the binding site domain of dynein in CRMP4 and its deletion. These constructs were used in the IP that was performed in the middle panel. Middle panel - Immunoprecipitation of DIC followed by western blot analysis of CRMP4 in COS7 cells overexpressing either GFP-CRMP4, or GFP-CRMP4 with deletion of amino acid 100-150, or GFP-CRMP4 overexpressing cells that were pre-incubated with a 10 µm mixture of peptides 1-4 (size of ~91 KDa). Lower panel - Western blot analysis of total protein levels before the pull-down assay (DIC size: ~75 KDa).

(C) Quantification of the Western blot in B from 3 independent repeats. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each technical repeat. One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.007, *p=0.0261.

(D) Upper panel - immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - total protein input (size of ~150 KDa).

(E) Quantification of the blot in D from 3 independent repeats. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, **p=0.01.

(F) Upper panel - Immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - Total input (size of ~150 KDa).

(G) Quantification of the blot in F. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0299.

(H) Immunoprecipitation of DIC (size of ~75 KDa) followed by Western blot analysis of CRMP4 (size of ~64 KDa) in COS7 cells that were transfected with CRMP4 and AAV9-50aa or its control. IgG antibody was used as a control.

(I) Quantification of the blot in H from 3 independent repeats. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0479.
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 5

Figure 5 -CRMP4-dynein interaction is enhanced in ALS motor neuron axons

(A) Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in SOD1G93A compared to WT P90 sciatic nerves under physiological conditions. IgG antibody was used as a control.

(B) Quantification of the blot in A. 3 repeats, 12 sciatic nerves per condition were used in each repeat. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Data presented as mean ±SE (Ratio Paired t-test, *p = 0.0416).

(C) Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in Cos7 cells that were transfected with mutant CRMP4 I141V compared to control. IgG antibody was used as a control.

(D) Quantification of four repeated pull down in C. The CRMP4 intensity band was normalized to the DIC intensity band for each repeat. (Student's t-test, n=4, Data presented as mean ±SE, *p=0.0393).

(E) Representative images from the proximity ligation assay (For explanation of PLA technique; please refer to method section) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment. Scale bar: 5µm.

(F) Quantification of the CRMP4-DIC puncta number per primary motor neuron axon in each condition. We analyzed ~20 axons per condition from 3 independent chambers per group (One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.01 *p=0.04)

(G) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human-derived proximal axons post peptides treatment, Sema3A treatment, Sema3A + peptides treatment or untreated controls. Scale bar: 5µm.

(H) Quantification of the CRMP4-DIC puncta number per axon in healthy or C9orf72 human-derived MN proximal axons after Sema3A treatment, Sema3A + peptides treatment or untreated controls. Data collected from 3 independent chambers in each condition. Total of 37 healthy untreated proximal axons, 61 healthy proximal axons with peptides treatment, 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment. 67 C9orf72 untreated proximal axons, 63 C9orf72 proximal axons with peptides treatment, 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored. Data presented as mean ±SE. One-way ANOVA, Tukey's multiple comparisons test, n=3, ****p<0.0001.
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 6

Figure 6 - Retrograde CRMP4-dynein complex formation mediates MN loss in ALS

(A) Representative images of CTX signal in healthy and C9orf72 human IPSC-derived MNs before and after Sema3A application. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 40 μm.

(B-C) Quantification of CTX signal in healthy and C9orf72 IPSC-derived MNs before and 3 days after applying Sema3A to distal compartment, compared with untreated control. 3 independent chambers in each condition were analyzed. Average of ~150 neurons per condition monitored. Student's t-test, n=3, Data presented as mean ±SE, *p<0.05.

(D) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A or untreated. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 30 μm.

(E) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A. 3 independent chambers in each condition were analyzed. ~200 neurons were monitored per each condition. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p<0.05, **p<0.01. Dynein inhibitor and Dynasore treatments were used as a negative control.

(F-G) Representative images and quantification of C9orf72 iPSC-derived MNs in the proximal compartment of an MFC before and after Sema3A treatment with and without 10 ug peptides 1-4. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. 3 independent chambers in each condition were analyzed. ~200 neurons per condition monitored. Scale bar: 40µm. One-way ANOVA Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, **p =0.004.
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 7

Figure 7 - Blocking the formation of the CRMP4-dynein complexes reduces motor neuron toxicity in vivo

(A) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 20 µm.

(B) Quantification of active caspase 3 positive cells in P90 WT and SOD1G93A SC. 3 different mice in each condition analyzed. We monitored active caspase 3 expression in total of 108 cells in WT SC and 123 cells in SOD1G93A. Student's t-test, n=3, Data presented as mean ±SE, ****p<0.0001.

(C) Representative images of P90 SOD1G93A mice SC cross sections that were injected with AAV9-GFP/AAV9-50aa-GFP. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 10 µm.

(D) Quantification of caspase 3 positive cells in P90 SOD1G93A mice SC cross sections that were injected with either AAV9-GFP or AAV9-50aa-GFP. Data collected from 3 different mice in each condition. We monitored active caspase 3 expression in total of 228 cells in in P90 SOD1G93A mice SC cross sections that were injected with AAV9-GFP and 179 cells P90 SOD1G93A mice SC cross sections that were injected with AAV9-50aa-GFP. Student's t-test, n=3, Data presented as mean ±SE, ** p=0.0019.

(E) Quantification of the number of NeuN positive cells in P90 SOD1G93A mice SC cross sections that were injected with either AAV9-GFP or AAV9-50aa-GFP. Data collected from 3 different mice in each condition. We monitored the number of NeuN positive cells from total of 228 cells in P90 SOD1G93A mice SC cross sections that were injected with AAV9-GFP and 179 cells P90 SOD1G93A mice SC cross sections that were injected with AAV9-50aa-GFP. Unpaired t test with Welch's correction, n=3, Data presented as mean ±SE, * p=0.0484.

(F) Working model - CRMP4-dynein complex formation is enhanced in ALS disease and leads to subtype-specific neuronal loss.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 1

Figure 1. DNMT level in the spinal motor neurons of SBMA mice.

(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice.

(B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3).

(C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3).

(D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group).

(F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse.

(G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice.

(H) Quantification of the signal intensity of Dnmt1 bands (n = 3).

Data information: One-way ANOVA with Tukey test (B,C,E). Unpaired t-test (H). Error bars, s.e.m. Scale bar, 20 µm (D,F). *P < 0.05. N.S.; not significant. 1C2, polyglutamine. DAPI, 4',6-diamidino-2-phenylindole. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 2

Figure 2. Dnmt1 suppression improves the cell viability of neuronal SBMA cells.

(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).

(B) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT-qPCR (n = 3).

(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.

(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).

(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).

(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).

(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.

(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).

Data information: Unpaired t-test. Error bars, s.e.m. *P < 0.05, N.S.; not significant. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 3

Figure 3. RG108 improves the motor function and survival of SBMA mice.

(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).

(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.

(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).

(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).

Data information: Two-way ANOVA with Tukey's test (A-C). Log-rank test (D). Unpaired t-test (G,I). Error bars, s.e.m. Scale bar, 20 μm (H). *P < 0.05. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 4

Figure 4. RG108 attenuates motor neuron degeneration in SBMA mice without degrading the disease-causing abnormal AR protein.

(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody.

(B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).

(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR.

(D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).

(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).

(F,G) Immunohistochemistry for ChAT (F) and quantitative analysis of the neuron sizes (G) in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).

(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).

Data information: Unpaired t-test. Error bars, s.e.m. Scale bar, 20 µm (A,F). *P < 0.05. N.S.; not significant. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 5

Figure 5. DNA methylation array analysis identified Hes5 as a key molecule for SBMA pathogenesis.

(A) Immunoblotting of SH-SY5Y cells stably expressing AR-24Q (SH24Q) or AR-97Q (SH97Q) for Dnmt1, Dnmt3a and Dnmt3b.

(B) Densitometry quantification showing that Dnmt1 level was up-regulated in SH97Q cells compared with SH24Q cells (n = 3).

(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).

(E) Seven genes with DNA hyper-methylation in their CpG islands were identified by DNA methylation array analysis.

(F) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).

(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).

(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.

(I) Hes5 immunoreactivity of spinal motor neurons in wild-type, AR-24Q, and AR-97Q mice (n = 3).

(J) Hes5 mRNA levels in the spinal cords of wild-type, AR-24Q, and AR-97Q mice (n = 3).

Data information: Unpaired t-test (B, D, F, G). One-way ANOVA with Tukey test (I, J). Error bars, s.e.m. Scale bar, 50 µm (I). *P < 0.05. N.S.; not significant. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 6

Figure 6. RG108 restores Hes5 level by reducing DNA methylation.

(A,B) DNA methylation levels of the HES5 promoter region (A) and the HES5 mRNA levels (n = 3) (B) of SH97Q cells treated with DHT and RG108.

(C,D) DNA methylation levels of the Hes5 promoter region (C) and the mRNA levels of Hes5 (n = 3) (D) in DHT-treated NSC97Q cells treated with or without RG108.

(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).

(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).

Data information: Unpaired t-test. Error bars, s.e.m. Scale bar, 20 µm (F). *P < 0.05. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 7

Figure 7. Hes5 copes with pathogenic AR cytotoxicity.

(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).

(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).

(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.

(D, E) Cell viability measured with the WST-8 assay (D), and immunoblotting (E) of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).

(F) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).

(G) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR.

(H) AR aggregation as quantified by densitometry (n = 3).

Data information: Unpaired t-test. Error bars, s.e.m. *P < 0.05. N.S.; not significant. The exact P value is in Appendix Table S3.
DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5 (2019)
Figure 8

Figure 8. Hes5 plays a protective role against SBMA pathomechanism through Smad2 activation.

(A) Immunoblots of NSC34 cells treated with Hes5 siRNA.

(B) Quantification of protein levels of pSmad2, Smad2, phospho-IkBα and heat shock factor-1 (Hsf1) (n = 3).

(C) siRNA-mediated knockdown of Hes5 down-regulated Hes5 protein level (n = 3).

(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).

(E) Protein levels of Smad2, pSmad2, and Hes5 in NSC97Q cells transfected with Hes5 vector (n = 3).

(F) Immunoblots of Smad2, pSmad2, and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q (n = 3).

(G) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q (n = 3).

(H) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q (n = 6).

(I) Western blots of Smad2, pSmad2, and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5-containing lentiviral vector.

Data information: Unpaired t-test. Error bars, s.e.m. *P < 0.05. N.S.; not significant. The exact P value is in Appendix Table S3.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 1

Figure 1: CHK1 is an essential survival factor for Hoxb8-FL cells.

(A) Hoxb8-FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1-inhibitors PF-477736 (PF) or CHIR-124 (CHIR). Representative histograms of CHK1i-treated (grey) or DMSO-treated (black) cells, analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown.

(B) WT Hoxb8-FL cells were treated with CHIR-124 in the presence or absence of the pan-caspase inhibitor QVD. Cell death was assessed after 24h using flow cytometry as in (A). (C) Western blot of CHIR-treated Hoxb8-FL WT and Bax-/-Bak-/- cells using the indicated antibodies.

(D) WT and Bax-/-Bak-/- Hoxb8-FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1-inhibition by real-time RT-qPCR. Bars represent means ± S.D. of independent experiments (n=4-6), performed in duplicates.

(E) 50.000 Hoxb8-FL cells of the indicated genotypes were seeded to assess cell growth in response to CHK1-inhibition. Cells were counted 48h post seeding.

Data information: Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test. Bars in (B) represent means ± S.E.M. (n=2 biological and technical replicates per genotype). Bars in (E) represent means ± S.E.M. (n=3 biological replicates per genotype).