EMBO/soda-nel huggingface dataset:
Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance (2017)
Figure 2

Figure 2. dog1 mutant plants are susceptible to drought stress.

A Col-0 (WT) and dog1-3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days. A picture of a representative experiment is shown. Scale bar, 2 cm.

B Quantification of recovered and dead Col-0 (WT), dog1-3 and dog1-4 plants; plants were scored as dead when unable to grow and produced offspring after an extended recovery period.

C Analysis of DOG1 sense expression in drought-treated vs. control Col-0 (WT) and dog1-3 mutant plants.

D dog1-3 mutant plants are defective in the regulation of the majority of the tested drought marker genes. Col-0 (WT) and dog1-3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT-qPCR. Error bars represent standard deviation, and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.
Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance (2017)
Figure 3

Figure 3. DOG1 upregulation by ABA is strongest 40 days after germination and requires asDOG1.

A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant.

B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm.

C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant.

D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm.

E RT-qPCR analysis of DOG1 sense mRNA level.

F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants.

G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA.

H Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.
Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance (2017)
Figure 4

Figure 4. High-level DOG1 expression due to antisense removal causes enhanced drought resistance in Arabidopsis.

A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm.

B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.
CARS senses cysteine deprivation to activate AMPK for cell survival (2021)
Figure 1

Figure 1 Cystine deprivation activates AMPK through CaMKK2

(A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates.

(B-D) 293T cells were treated with cystine-deficient medium for 0, 0.5, 1, 2, 4, or 8 h (B) or treated with medium containing 200 µM, 100 µM, 50 µM, 25 µM or 0 µM cystine for 8 h (C); RCC4, SK-MES, SK-hep-1 or Hep3B cells were treated with cystine-deficient medium for indicated time (D). Protein levels of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor were measured by WB, and actin served as the loading control.

(E) A schematic diagram of the hypothetical mechanism showing how cystine deprivation induces AMPK activation.

(F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control.

(G) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells and RCC4 cells treated with 1 µg/ml STO-609 or DMSO for 8 h during cystine deprivation. Actin served as the loading control.

(H) 293T cells were transfected with HA-AMPKα1 alone or together with Flag-CaMKK2 for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and then, WB analysis was performed.

(I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.
CARS senses cysteine deprivation to activate AMPK for cell survival (2021)
Figure 2

Figure 2 CARS is critical for AMPK activation under cystine deprivation conditions

(A) Venn diagram showing the overlap of CaMKK2-binding proteins during cystine deprivation and cyst(e)ine metabolism-related proteins. The schematic diagram shows the peptides detected in the results of IP-MS assays of the AHCY and CARS proteins. The CaMKK2-binding proteins are listed in the Table EV3 and the cyst(e)ine metabolism-related proteins are listed in the Table EV4.

(B) 293T cells were transfected with HA-CaMKK2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.

(C) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-CaMKK2, followed by WB analysis with anti-CaMKK2 and anti-CARS.

(D) Flag pull-down assays were performed with Flag-EV or Flag-fused CaMKK2 protein and His-CARS purified from E. coli.

(E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control.

(F) 293T cells were first transfected with shRNAs targeting CARS or NTC, then transfected with HA-AMPKα1 or HA-AMPKα1 and Flag-CaMKK2 for 48 h, and then, they were cultured with cystine-deficient medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag and subjected to Western blot analysis.

(G) 293T cells were transfected with shRNAs targeting CARS or NTC, then they were cultured with cystine-deficient medium for 8 h. Cell lysates were immunoprecipitated with anti-AMPKα and subjected to WB analysis with anti-AMPKα and anti-CaMKK2.

(H) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, CARS protein expression in 293T cells and RCC4 cells overexpressing Flag-CARS. Actin served as the loading control.

(I) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells were further transfected with shRNAs targeting LKB1, CaMKK2, or TAK1. Actin served as the loading control.

(J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.
CARS senses cysteine deprivation to activate AMPK for cell survival (2021)
Figure 3

Figure 3 CARS activates AMPK by sensing cystine deficiency upon binding to AMPKγ2

(A) His pull-down assay was performed with His-EV- or His-fused CARS protein and HA-tagged AMPK subunit proteins overexpressed in 293T cells.

(B, C) GST pull-down assay was performed with GST-EV, GST-AMPKγ1, GST-AMPKγ2 (B) or GST-EV, GST-AMPKγ2, GST-AMPKγ2-N, GST-AMPKγ2-C (C) proteins and His-tagged CARS protein purified from E. coli. The segments of AMPKγ2 are shown in the bar graph above (C). The red arrows indicate the target bands.

(D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control.

(F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.

(G) 293T cells were transfected with HA-AMPKγ2 for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-CARS, followed by WB analysis with anti-HA and anti-CARS.

(H) GST pull-down of His-CARS by GST-AMPKγ2 using protein purified from E. coli, followed by WB analysis. Cysteine (0 μM, 2 μM, 10 μM, 50 μM and 250 μM) was added to the experimental buffer.

(I) The schematic diagram shows that cystine deficiency-induced AMPK activation is mediated by CaMKK2, which is recruited by CARS and the AMPKγ2 complex.
CARS senses cysteine deprivation to activate AMPK for cell survival (2021)
Figure 4

Figure 4 The CARS-CaMKK2-AMPK axis promotes cell survival when cystine is eliminated

(A) The crystal violet assay was performed in cystine-deficient medium-cultured 293T, RCC4 and SK-MES cells with or without 1 µg/ml STO-609 for 24 h.

(B) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.

(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.

(E) WB analysis of CARS, CaMKK2 and AMPKγ2 protein expression in RCC4, SK-MES, 293T, 786-O, NCI-1650, and HT-1080 cells. Actin served as the loading control.

(F, G) WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression (F) and a crystal violet assay were performed (G, left) with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. After scanning, 20% glacial acetic acid was added to dissolve the crystal violet, and the absorbance of each well was measured at 570 nm (OD570) to calculate relative cell viability (G, right). Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 1

Figure 1. SNP rs4971059 Locates in an Active Enhancer at the 6th Intron of TRIM46.

A. Physical map of the locus of TRIM46 [chromosome 1: 155173381-155184971 (hg38)] and SNP rs4971059 [chromosome 1: 155176305 (hg38)].

B. HaploReg v4.1 was used to predict the potential functions of SNP rs4971059 (left). The yellow box means "Weak Enhancer 2" (H3K4me1 and DNase enrichment), the orange box means "H3K4me1 Enhancer", and the black box means "missing data". ChIP assays were performed with antibodies against H3K4me1, H3K4me3, or H3K27Ac in the region surrounding SNP rs4971059 (right). C. Luciferase assays showing enhancer activity in the SNP rs4971059 region in MCF-7 cells. MCF-7 cells were transfected with the indicated promoters: PGL3-SV40, PGL3-SV40-SNP[G] or PGL3-SV40-SNP[A] (left); PGL3-TRIM46, PGL3-TRIM46-SNP[G] or PGL3-TRIM46-SNP[A] (right). Forty-eight hours after the transfection, luciferase activity was measured. Relative luciferase activity was calculated as firefly luciferase activity divided by renilla luciferase activity and shown relative to the control.

D. The SNP rs4971059-containing region formed close contact with the promoter of TRIM46 in a 3C assay. DpnII fragments in 1q22 were shown on the top panel (hg38). SNP rs4971059 resides in 3C fragment 17, and fragment 5 is in the genomic region 3-kb upstream of the TSS of TRIM46. PCR primers were designed near the end of DpnII fragments in the same orientation and 3C assays were performed with a constant primer in fragment 17 (lower-left panel) or a constant primer in fragment 5 (lower-right panel).

E. eQTL analysis demonstrating the correlation between rs4971059 genotype and expression of TRIM46. The panel showed results from breast cancer tissues obtained from the mastectomy of the patients. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively (FDR=0.00147).

F. A physical 650-kb window of the genomic distribution of genes surrounding SNP rs4971059, such as EFNA4, KRTCAP2, MUC1, MIR92B, THBS3,and DAP3 (top). Total RNAs were extracted from [GG], [GA], or [AA] MCF-10A cells and analyzed for the expression of EFNA4, KRTCAP2, TRIM46, MUC1, THBS3, MIR92B, or DAP3 by RT-qPCR. TRIM28 was examined as a negative control (bottom).

Data information: In (B, C, D, and F), each bar represents the mean ± SD for biological triplicate experiments (*P<0.05, two-tailed unpaired t-test).
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 2

Figure 2. TRIM46 Is Physically Associated with HDAC1.

A. Mass spectrometry analysis of TRIM46-associated proteins. Whole-cell extracts from MCF-7 cells stably expressing FLAG-TRIM46 were subjected to affinity purification with anti-FLAG immobilized on the agarose beads. The purified protein complex was resolved on SDS-PAGE and silver-stained. The bands were retrieved and analyzed by mass spectrometry.

B. MCF-7 cells were transfected with a control vector or FLAG-TRIM46 for 40 h and treated with DMSO or MG132 (2μM) for 10h. Cellular lysates were then collected for western blotting with antibodies against the indicated proteins.

C. MCF-7 cells were transfected with control vector or FLAG-TRIM46 for 48 h. Whole-cell lysates were prepared, and immunoprecipitation was performed with anti-FLAG followed by immunoblotting with antibodies against indicated proteins (top). Immunoprecipitation assays in MCF-7 cells with anti-TRIM46 followed by immunoblotting with antibodies against the indicated proteins (bottom).

D. Schematic diagrams of TRIM46 deletion mutants (left). Coomassie brilliant blue (CBB) staining of GST-fused proteins as indicated (right). GST-FL was shown with the arrow.

E. GST pull-down experiments were performed with bacterially expressed GST-fused proteins as shown and in vitro transcribed/translated full-length HDAC1.

F. Schematic diagrams of HDAC1 deletion mutants. GST pull-down experiments were performed with bacterially expressed GST-fused proteins as indicated and in vitro transcribed/translated TRIM46.
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 3

Figure 3. TRIM46 Negatively Regulates HDAC1.

A. TRIM46 negatively regulates the steady-state level of the HDAC1 protein. MCF-7 cells were transfected with increasing amounts of FLAG-TRIM46. 48 hours after transfection, cells were treated with DMSO or MG132 for 10 h before cells were collected for western blotting (left). Alternatively, total RNAs were extracted and analyzed for HDAC1 expression by RT-qPCR (right).

B. Cellular proteins were extracted from [GG], [GA], or [AA] MCF-7 cells, and western blotting was performed with polyclonal antibodies against TRIM46, HDAC1, and GAPDH.

C. Cellular proteins were extracted from [GG], [GA], or [AA] MCF-10A cells, and western blotting was performed with polyclonal antibodies against TRIM46, HDAC1, or GAPDH.

D. Total RNAs were extracted from [GG], [GA], or [AA] MCF-10A cells and analyzed for HDAC1 expression by RT-qPCR.

E. MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or a siRNA-resistant TRIM46 construct together with TRIM46 siRNA. Western blotting (left) and RT-qPCR (right) were performed as in A.

F. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1.

G. MCF-7 cells were treated as described in F, except that MG132 (10 μM) was added to cells 10 h before cellular proteins were collected for western blotting (left). Quantitation was performed on the right panel as in F.

H. MCF-7 cells were treated with control siRNA or TRIM46 siRNA. Forty-eight hours after transfection, cells were treated with 50 μg/mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis (left). Quantitation was performed on the right panel as in F.

Data information: In (A, D, E, F, G, and H), each bar represents the mean ± SD for biological triplicate experiments (*P<0.05, **P<0.01, two-tailed unpaired t-test).
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 4

Figure 4. TRIM46 Promotes HDAC1 Ubiquitination and Degradation.

A. In vitro ubiquitination assays with GST-HDAC1, FLAG-TRIM46, FLAG-TRIM46ΔRING, or FLAG-TRIM46ΔC in the presence or absence of ubiquitin and E2. HDAC1 was detected using western blotting.

B-D. In vivo ubiquitination assays performed in MCF-7 cells. (B) Stable shCTR- or shTRIM46-expressing MCF7 cells were transfected with His-Ub for 40 h. Cells were then treated with MG132 (10 μM) for 12 h before Ni-NTA bead precipitation followed by IB with anti-HDAC1. (C) and (D) MCF-7 cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni-NTA bead precipitation followed by IB with anti-HDAC1.

E. MCF-7 cells were co-transfected with Myc-TRIM46 and FLAG-tagged wild-type HDAC1 or HDAC1 deletion mutants for 48 h. Cellular proteins were extracted for western blotting analysis.

F. MCF-7 cells were co-transfected with Myc-TRIM46, HA-ubiquitin, and FLAG-tagged wild-type HDAC1 or HDAC1 mutants for 40 h. Cells were treated with MG132 for 12 h before cellular extracts were prepared for immunoprecipitation assays with anti-FLAG followed by immunoblotting with anti-ubiquitin.

G. MCF-7 cells were co-transfected with FLAG-HDAC1-K123R, K126R, K143R, or K144R along with indicated plasmids for 40 h. Cells were treated with MG132 (10 μM) for 10 h before cellular extracts were prepared for Ni-NTA bead precipitation.

H. MCF-7 cells were co-transfected with Myc-TRIM46 and wild-type HDAC1 or indicated HDAC1 mutants for 48 h. Cellular proteins were extracted for western blotting analysis.
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 5

Figure 5. The TRIM46-HDAC1 Axis Intersects with DNA Replication and Repair Pathways.

A. ChIP-seq analysis was performed in control or TRIM46-overexpressing MCF-7 cells with antibodies against HDAC1. Density distributions (read count per million of mapped reads) of HDAC1 peaks were analyzed by deepTools.

B. Scatter plot of RNA-Seq data comparing genes between control and TRIM46-depleted MCF-7 cells. The clustering of the 630 differentially expressed genes is shown. RNA-seq experiments were performed in duplicate, and overlapping genes were analyzed.

C. The Venn diagram of overlapping target genes of the TRIM46-HDAC1 axis detected by ChIP-seq and RNA-seq.

D. Classification of genes targeted by the TRIM46-HDAC1 axis using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

E-G. Verification of representative genes targeted by the TRIM46-HDAC1 axis. (E) MCF-7 cells were transfected with empty vector, FLAG-TRIM46, or FLAG-TRIM46 together with FLAG-HDAC1 for 48 h. (F) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or TRIM46 siRNA together with HDAC1 siRNA for 72 h. (G) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or a siRNA-resistant TRIM46 construct together with TRIM46 siRNA for 72 h. Total RNAs were extracted from cells and RT-qPCR was performed with primers specific for indicted genes.

Data information: In (E-G), each bar represents the mean ± SD for triplicate experiments (*P<0.05, two-tailed unpaired t-test).
SNP rs4971059 Predisposes to Breast Carcinogenesis and Chemoresistance via TRIM46-mediated HDAC1 degradation (2021)
Figure 6

Figure 6. The TRIM46-HDAC1 Pathway Functions in DNA Replication and Repair in Breast Cancer Cells.

A. MCF-7 cells treated with control siRNA, TRIM46 siRNA, or TRIM46 siRNA and HDAC1 siRNA were synchronized by a double thymidine block and released for 0 h, 12 h, 30 h, 36 h, or 42 h, respectively. Cell cycle progression after releasing was analyzed by flow cytometry.

B. EdU assays were performed in MCF-7 cells treated with control siRNA, TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h, or MCF-10A cells treated with vector, FLAG-TRIM46 or FLAG-TRIM46 and FLAG-HDAC1 for 48 h. Representative images from FACS analysis are shown on the left panel, the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel.

C. Western blotting analysis of TRIM46 protein expression in different breast cell lines.

D. ssDNA formation in siRNA-treated MCF-7 cells after HU treatment. BrdU (20 μg/ml) was added during the last 24 h of siRNA treatment and was removed by a brief wash prior to HU treatment (4 mM, 2 h), pre-extraction, and fixation. BrdU in ssDNA patches and PCNA were detected without a DNA denaturation step. Scale bar, 10 μm. Diagrams show the frequency of PCNA-positive BrdU foci.

E. DNA fiber analysis of the rate of replication elongation. SiRNA-treated MCF-7 cells were sequentially pulse-labeled with 50 μM IdU and 50 μM CldU, each for 30 min. A sketch delineating experimental design and representative images of dual-labeled fibers are shown. Bars represent the median CldU tract length (n = 200 in each group). ****P < 0.0001, two-tailed unpaired t-test.

F. MCF-7 cells treated with control siRNA, TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA were treated with 4 μM cisplatin for 48 h, and then double-stained with annexin V and propidium iodide. Cell apoptosis was determined by flow cytometry. Representative images from FACS analysis are shown on the left panel, the percentage of apoptotic cells was quantified by FlowJo software on the right panel.

G. Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation. MCF-7 cells were treated with 4 μM cisplatin for 8 h before harvesting. Cell lysates were analyzed by western blotting.

Data information: In (B, D, and F), each bar represents the mean ± SD for biological triplicate experiments (*P<0.05, **P < 0.01, two-tailed unpaired t-test).