EMBO/soda-nel huggingface dataset:
Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways. (2018)
Figure 3

Figure 3. Knockdown of ESRP1 does not induce EMT in models of resistance to endocrine therapy.

A Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC2 model-2-control and 2C3 cells. Scale bar: 50 µm.

B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm.

C-D Protein expression of key EMT-TFs in ESRP1 knockdown cells (2C3 and 9C2) compared to endocrine resistant control cell lines (2-control and 9-control) using Western blotting analysis.

E Protein expression of key EMT-TFs in ESRP1 knockdown cells (T-47D-kESRP1) and ESRP1-overexpressing MCF-7 cells compared to their control counterparts (T-47D-control and MCF-7-control, respectively).
Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways. (2018)
Figure 4

Figure 4. A unique set of EMT genes are altered at the splicing level but not at the gene level.

A Doughnut chart for RNA-seq analysis showing the differentially regulated alternative splicing events (ASEs) and their corresponding genes in ESRP1 knockdown cells compared to control cells; SE: skipped exon, RI: retained intron, MXE: mutually exclusive exons, A5SS: alternative 5' splice site, and A3SS: alternative 3' splice site). The "percent spliced in" (PSI or ψ) value was estimated. Differences in inclusion levels (∆ψ) between samples and their significance were calculated (P<0.05 and IΔψI>0.1). Circle 1: all ASEs; Circle 2: all genes corresponding to all ASEs; Circle

3: significant ASEs; Circle 4: significant genes corresponding to significantly altered ASEs; Circle 5: significant ASEs with ESRP1 motifs; Circle 6: significant genes corresponding to significantly altered ASEs with ESRP1 motifs. ESRP-binding motifs were obtained from Dittmar et al.31[] and analyzed based on the following assumption: the presence of the motif in the downstream region of an exon results in inclusion of the exon, while upstream or intraexonic location leads to exclusion of the exon.

B Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC2 set).

C Radar plot showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC2 set).

D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC9 set).

E Radar plots showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC9 set).
Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways. (2018)
Figure 7

Figure 7. Impact of ESRP1 knockdown on endocrine resistant breast cancer.

A Volcano plots showing the distribution of significant gene expression changes using the HTA 2.0 platform.

B Differential gene expression analysis of ESRP1 knockdown compared to resistant control cells using HTA 2.0 platform; downregulated genes in ESRP1 knockdown (left panel) and upregulated genes in ESRP1 knockdown (right panel) common in 2-control versus 2C3, and 9-control versus 9C2.

C Validation of the protein levels of FASN, SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis. GAPDH was used as the loading control. The data are representative of three individual biological replicates.

Expanded View Figure Legends
Intra-arterial transplantation of HLA-matched donor mesoangioblasts in Duchenne Muscular Dystrophy (2015)
Figure 6

Figure 6: Effects of MAB treatment on the immune response of DMD patients.

Panel A: viral and donor specific T-cell responses were measured by IFN-γ ELISpot at different time points: before the beginning of treatment (PRE), prior to each infusion and 2 months after the fourth infusion. Infusions are indicated by arrows. Patient’s peripheral blood mononuclear cells (PBMC) were challenged with irradiated autologous lymphoblastoid cell lines (EBV line host), with cytomegalovirus glycin extract (CMV antigen)(Gehrz et al, 1987) and with the following irradiated cells harvested from the donor: untreated mesoangioblasts (MAB donor), MAB activated by 48 hour-exposure to 500 IU/mL IFN-γ (γMAB donor), myotubes differentiated from MAB (myotubes donor) and PBMC (PBMC donor). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. Donor PBMC were challenged with autologous targets as negative controls. Results are expressed as number of specific cells/105 PBMC and calculated according to the following formula: number of spots produced by patient’s PBMC – number of spots produced by donor’s PBMC. Results for patients Pt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.

Panel B: dystrophin specific T-cell responses were measured by IFN-γ ELISpot at two time points: before the beginning of treatment (PRE, white bar) and 2 months after the fourth infusion (black bar). PBMC were challenged with a library of 15-mer overlapping peptides covering the protein sequence (pool 1-8). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. The grey area represents the threshold calculated on a cohort of healthy donors. Results for Pt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.
Neuropilin-1 expression distinguishes self-reactive helper T cells in systemic autoimmune disease (2022)
Figure 3

Figure 3 Humoral responses against exogenous antigens are independent of Nr4a2

(A-C) Young Male BXSB.Yaa and BXSB.Yaa Nr4a2 cKO mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of antibodies binding NP-31 (left) and NP-4 (right) epitopes by ELISA across a range of diluted serum samples with IgG1-specifc (A) and IgG2a-specific antibodies (B). The ratio for low to high affinity (NP-4:NP-31) antibodies was calculated for IgG2a (left) and IgG1 (right) antibodies (C). Error bars, SEM, n=5 mice per group; p>0.1 two-way ANOVA.

(D-F) Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).

(G) Stool samples from Young Male BXSB.Yaa and BXSB.Yaa Nr4a2 cKO mice were measured for IgA concentration by ELISA. Points are samples from individual mice, error bars, SEM, n=8 from 2 independent experiments; p>0.1 Unpaired Student's T test.
Neuropilin-1 expression distinguishes self-reactive helper T cells in systemic autoimmune disease (2022)
Figure 8

Figure 8 NRP1-expressing Th cells are upregulated in human SLE patients

(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)

(B-D) NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) and CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.

(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.

(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.

Extended View Figure Legends
FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers (2022)
Figure 2

Figure 2. Inhibition of FOXM1 increases ROS and induces NET formation.

A. Representative images of human neutrophils treated with the FOXM1 inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red. Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).

B. Quantification of ROS levels in human neutrophils treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).

C. Quantification of NET formation in human neutrophils treated with FDI-6 or in combination with NAC. PMA treatment served as a positive control. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).
FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers (2022)
Figure 4

Figure 4. Activation of TREM1 enhances wound healing and increases FOXM1+ neutrophils in the wounds of diabetic mice.

A. Representative images of wounded skin after topical treatment with either vehicle (IgG isotype control) or ⍺-TREM1 activator at 0, 2, 4, 6, and 8 days after wounding. Percent of wound area at each time following vehicle or ⍺-TREM1 activator treatment relative to the original wound area. Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺-TREM1 treated wounds were performed with Fiji software. Data presented as mean ± SD. **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey's post-hoc test).

B. Representative pictures of vehicle (IgG isotype control) and ⍺-TREM1 treated diabetic wounds at day 4 show basal keratin marker K5, and neutrophil marker Ly6G, FOXM1 and citH3. Treatment of wounds with ⍺-TREM1 resulted in increased FOXM1+ and decreased citH3+ neutrophils compared to vehicle treated diabetic wounds. (Scale bar: 50µm). Quantification of mean fluorescence intensity was performed with Fiji software. n= 7 diabetic vehicle wounds and 6 diabetic ⍺-TREM1 wounds. Data presented as mean ± SD. *P < 0.05, *P < 0.05, and **P < 0.01 (two-tailed unpaired Student's t-test).
FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers (2022)
Figure 5

Figure 5. TREM1 expression is associated with the clinical outcome of healing in DFUs.

A. qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs (n = 5 healing and n= 6 nonhealing). Data presented as mean ± SD. *P < 0.05. **P < 0.01 (two-tailed unpaired Student's t-test).

B. Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing (n=4) and nonhealing (n=4) is shown in the graph. Data presented as mean ± SEM. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).

C. Representative images of cit-H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs, which was confirmed by corresponding quantification from healing (n=3) and nonhealing (n=3), shown in the graph. Data presented as mean ± SD. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).

Expanded View Figure Legends
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 1

Figure 1. Syt11 knockdown accelerates exo-endocytosis and the subsequent vesicle replenishment

A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments.

B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.

D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse.

E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons.

F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRG neurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.

I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization.

J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.

All data are presented as mean ± s.e.m. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 2

Figure 2. Both stimulus-coupled and constitutive endocytosis are accelerated by Syt11 knockdown

A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.

C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.

D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm.

E. Quantification of constitutive FM 1-43 uptake with different loading times.

F. Basal [Ca2+]i in DRG neurons measured with Fura-2.

G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.

H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.

I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).

All data are presented as mean ± s.e.m. of 3 to 4 independent experiments. Kolmogorov-Smirnov test for (B) and (G), one-way ANOVA for (I), Student’s t-test for (C), (E), and (F), *P < 0.05, **P < 0.01, ***P < 0.001.
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 4

Figure 4. Syt11 localizes to vesicle recycling pathways

A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm.

B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 5

Figure 5. Syt11 inhibits clathrin-mediated endocytosis

A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm.

B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm.

C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).

D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C).

E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.

All data are presented as mean ± s.e.m. of 4 to 5 independent experiments. One-way ANOVA for (A) and (B), Student’s t-test for (D), (E), and (G), **P < 0.01, ***P < 0.001; NS, not significant.
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 6

Figure 6. Syt11 knockdown increases the frequency of bulk endocytosis

A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right.

C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation.

E, F. Size and duration of Cm decay during bulk endocytosis.

G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.

All data are presented as mean ± s.e.m. of 3 (H) or 6 (A-F) independent experiments. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 7

Figure 7. Vesicle size remains unaffected in Syt11-knockdown neurons

A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.

B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function.

D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).

Data are presented as mean ± s.e.m. of 3 independent experiments. ANOVA, P = 0.995 for (D) and P = 0.998 for (E).
Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis (2015)
Figure 9

Figure 9. Functional domains of Syt11 in endocytosis

A. Diagram of the mutant forms of Syt11 used for rescue experiments.

B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.

All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student’s t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 1

Figure 1. CHK2 is involved in oxidative stress induced autophagy

(A) Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3 in H1299 cells in normal medium (HBSS ''-'') or after 1 hr HBSS starvation (HBSS ''+'') pretreated with or without NAC (3mM).

(B) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment.

(C) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid.

(D) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably-expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500μM) treatment for 3h. Scale bar, 10 μm.

(E) Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500μM) treatment for 1h (n=30). Data are presented as mean±s.e.m. from 3 independent experiments; **P < 0.01 (Student's t-test).

(F) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures.

(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).
ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress (2020)
Figure 2

Figure 2. Oxidative stress enhances CHK2-Beclin 1 interaction

(A) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy-related genes in HCT116 cells. Lysates were extracted for immunoprecipitation with CHK2-specific antibody or control IgG, followed by probing with antibodies specific for Ulk1, Atg5, Beclin1, Atg7 or LC3.

(B) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after HBSS starvation in the cytoplasmic fractions.

(C) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after H2O2 (500μM) stimulation in the cytoplasmic fractions.

(D) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.

(E-F) HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation (E) or H2O2 (500μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.

(G-I) HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated or untreated (G) with HBSS starvation (H) or H2O2 (500μM) stimulation (I) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.

(J) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 hr.

(K) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 (500μM) stimulation. Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3.