EMBO/SourceData huggingface dataset:
Figure 2(C and D) Immunostaining assay of MAP2 (green) in control and SOX1KO cells on day 35 of dorsal neural differentiation (C), and quantiﬁcation of MAP2+ cells (D), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).(F and G) Immunostaining assay of GABA (green) in control and SOX1KO cells on day 35 of ventral neural differentiation (F), and quantiﬁcation of GABA+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).(H and I) Immunostaining assay of MAP2 (green) after SOX1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001 (V1-PAKI), p=0.0001 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).(J and K) Immunostaining assay of GABA (green) after SOX1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (J), and quantiﬁcation of GABA+ cells (K), p=0.0001 (V1-PAKI), p=0.0002 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).
Figure 3(A and B) Enrichment of H3K14Ac (left) and H3K27Ac (right) on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (A), p=0.0046 (H3K14Ac), p=0.0223 (H3K27Ac) and ventral neural differentiation (B), p=0.0499 (H3K14Ac), p=0.0022 (H3K27Ac). Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01 versus Ctrl group (unpaired two-tailed Student's t-test).(C Immunostaining assay of SOX1 (red) after 4 HDAC inhibitors (4i), including RG2833 (2833, 2μM), CAY10683 (CAY, 2μM), PCI-34051 (34051, 5μM) and Bufexamac (Buf, 12.5μM), treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI cells on day 16 of dorsal neural differentiation Scale bar, 100 μm.(G and H) Immunostaining assay of SOX1 (red) after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).(J and K) Immunostaining assay of MAP2 (green) after Bufexamac treatment in V1-PAKI cells on day 35 of dorsal neural differentiation (J), and quantiﬁcation of MAP2+ cells (K), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group (one-way ANOVA).(L) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) neural differentiation. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).
Figure 4(B and C) RIP analysis for endogenous interaction between HDAC10 and SOX1-OT V1 on day 16 of dorsal neural differentiation (B), p=0.0085 and ventral neural differentiation (C), p=0.0152. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus IgG group (unpaired two-tailed Student's t-test).(D and E) Enrichment of HDAC10 on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (D), p=0.0052 and ventral neural differentiation (E), p=0.0001. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).(G and H) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural differentiation (I), and quantiﬁcation of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (one-way ANOVA).
Figure 5(A and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm. (C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).(E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).
Figure 6(A and B) Immunostaining assay of SOX1 (red) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal (A) and ventral (B) organoids differentiation. Scale bar, 100 μm. (C) Immunostaining assay of MAP2 (green) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (D) Immunostaining assay of GABA (green) in control, V1-PAKI and SOX1KO cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.(E and F) Immunostaining assay of SOX1 (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm. (G) Immunostaining assay of MAP2 (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (H) Immunostaining assay of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.
Figure 1B) Total T cells were enriched from spleens of Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl mice. Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates, Gapdh used as loading control.CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Tumour growth in individual mice is shown for the indicated genotypes and treatments; number of mice eradicating the tumour is shown within the graphs (C).CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Survival curves are shown (D).Tumour growth in individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype; number of mice eradicating the tumour is shown within the graphs (G).individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm and control mice is shown (H).I, J) 10-12 days following MC38 tumour inoculation and treatment with anti-PD-1 antibody or isotype control, CD4cre Ptpn6/11wt/wt and CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm mice were sacrificed, and TILs analysed. Graphs depict frequencies of CD8+ T cells (gated as CD45+ TCRβ+ CD8+) expressing IFN-γ and TNFα upon re-stimulation (G) and percentages of CD8+ T cells (H).
Figure 2B) Ptpn6 and Ptpn11 copy number, as estimated through high resolution mass spectrometry in GzmBcre Ptpn6/11fl/fl and control CTLs.C and D) GzmBcre R26R-YFP reporter mice were injected subcutaneously with MC38 cells and analysed after 11 days. Graphs show the percentage of YFP+ cells among CD45+ CD8+ TILs (C) and the percentage of PD-1+ cells among CD45+ CD8+ YFP+ TILs (D). Results depict n = 4 mice (C and D).GzmBcre Ptpn6/11fl/fl and control Ptpn6/11fl/fl mice were subcutaneously injected with MC38 cells. Survival curves and statistical comparisons between different groups are shown (E).The tumour start depicts the time (days) following tumour engraftment after which the tumour is palpable, while tumour endpoint depicts the time (days) from tumour start until the reaching the maximal allowed tumour size (F).
Figure 3C) Numbers of CTLs (gated on CD8+ T cells) from Ptpn6/11fl/fl or GzmBcre Ptpn6/11fl/fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells.D) Quantitative high-resolution mass spectrometry was used to resolve the proteome of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (day 7). The plot depicts protein copy number in control and GzmBcre Ptpn6/11fl/fl cells. Significantly altered proteins are indicated in red; selected examples are annotated. Results are based on n = 3 biological replicates and two-tailed, unequal-variance t-test on log10 transformed copy number per cell values was used to compare differences between experimental groups.G) Graph illustrates the percentage of DAPI+ dead cells of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (gated on CD8+ T cells) at the indicated days as measured by flow cytometry.H) Protein copy number of Tnfrsf1b and Mcl-1 in GzmB cre Ptpn6/11fl/fl and control CTLs, as estimated through high resolution mass spectrometry.