EMBO/soda-nel huggingface dataset:
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 4

Figure 4 - Munc13 catalyzes opening of the Syx1 linker region via binding to the Munc18-1 domain 3a.

A Schematic depiction of smFRET assay. Positions of FRET label pair (S95C/S171C) in Syx1 are indicated as yellow pentagrams. 6×His-tagged Syx1 bound to Munc18-1 was immobilized on coverslip coated with PEG/PEG-NTA-Co2+. Syx1 linker region is colored by black.

B Example histograms of smFRET efficiency of Syx1 in complex with Munc18-1 WT in the absence and presence of the MUN domain, and within the SNARE complex.

C−E Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 QKAA (C), KKEE (D), and L348R (E) in the absence or presence of the MUN domain. The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain.

F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C−E.

G Effects of QKAA, KKEE, and L348R mutations on the interaction between the Munc18-1/Syx1 complex and the MUN domain by GST pull-down assay combined with immunoblotting. Results were shown with 6×His-tag antibody and GST-tag antibody.

Data information: In F, data are presented as mean values ± SEM, n.s., not significant; *, p < 0.05; ***, p < 0.001, n = 3 technical replicates. Statistical significance was analyzed by Student's t-test. In G, a representative gel displayed is from one of three independent replicates.
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 5

Figure 5 - Munc13 promotes Syb2 binding to the Munc18-1/Syx1 complex.

A Schematic depiction of Syb2 binding to Munc18-1/Syx1 complex in the presence of MUN domain. The extended instead of bent conformation of domain 3a within the Munc18-1/Syx1 complex is compatible with Syb2 binding.

B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks.

C Quantification of the normalized binding of Syb2 to Munc18-1/Syx1 complex. Integrated intensities of Munc18-1 bands were quantified. QKAA and L348R, but not KKEE, disrupted Syb2 binding to the Munc18-1/Syx1 complex.

Data information: In B, a representative gel displayed is from one of three independent replicates. In C, data are presented as mean values ± SD, n = 3 technical replicates.
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 6

Figure 6 - Potential interplay between the two conformational changes of Munc18-1 domain 3a and Syx1 linker region.

A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.

B Quantification of the FRET results in A.

C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain.

D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C.

Data information: In B data are presented as mean values ± SD, n = 3 technical replicates. In D, data are presented as mean values ± SEM, *, p < 0.05; **, p < 0.01, n = 3 technical replicates. Statistical significance was analyzed by Student's t-test.
Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly (2020)
Figure 8

Figure 8 - The Munc18-1 domain 3a constitutes a minimal template for Syx1 and Syb2.

A Schematic depiction of a putatively tertiary Munc18-1/Habc/H3 complex.

B Munc18-1, Habc and H3 construct a tertiary complex.

C The tertiary complex globally resembles the Munc18-1/Syx1 complex.

D Formation of the tertiary complex with Munc18-1 QKAA, KKEE, and L348R mutants. KKEE strongly impaired formation of the tertiary complex.

E Munc18-1 displays interaction with Syx1-Habc.

F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc.

G Cartoon illustration of the templating mechanism of domain 3a. The extended domain 3a (H11 and H12) binds to Syx1-H3 and Syb2 simultaneously. Color codes are as follows: Munc18-1, light gray; Munc18-1 H11 and H12, cyan; Syx1-H3, blue; Syb2, red. Key binding sites (L348, K332, and K333) on the H12 are highlighted with orange spheres.

Data information: All the proteins or protein complexes were analyzed on a Superdex 200 Increase 10/300 GL size-exclusion column.
Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer (2021)
Figure 1

Figure 1. A synergistc drug combination conditionally activated by mutant KRAS

A, Schematic of chemical synthetic vulnerability screens. Clinical related drugs (n=21) were combined with BI2536 to treat BEAS-2B-KRAS cells harboring a KRASG12V allele. Cell viability was determined 72 h post-treatment, with combination index (CI) calculated by CalcuSyn software: CI＞1, antagonism; 1＞CI＞0.8, synergy; CI＜0.8, strong synergy Drug combinations leading to strong synergy (CI＜1) in BEAS-2B-KRAS were further tested in KRAS wild-type BEAS-2B cells, where CI＜1 indicates toxicity and CI＞1 safety.

B, The Fa-CI plot of drug combinations (candidate drug plus BI2536) in KRAS-mutant (BEAS-2B-KRAS) and wild-type (BEAS-2B) cells. The data are mean values of two independent experiments (n=2) where all drugs are used at their IC50 determined in BEAS-2B-KRAS cells. Note that combination treatment with AZD4547 and BI2536 exhibit the leading therapeutic efficacy and safety profile, manifested by the greatest synergy (CI=0.25) in BEAS-2B-KRAS while the two drugs are antagonistic in BEAS-2B cells (CI>1).

C, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD

D, BEAS-2B and BEAS-2B-KRAS treated with the indicated drugs, alone or combination for 72 hours, were cultured in drug-free medium for additional 7-21 days. Surviving cells after the treatment were fixed and visualized by crystal violet staining.

E, Apoptotic assay of BEAS-2B and BEAS-2B-KRAS cells treated with vehicle (DMSO), AZD4547 (2.5 µM) and BI2536 (5 nM), alone or in combination for 72h. Data are presented as mean ± SD (n=3). *p<0.05 and ***P<0.001 by two-way ANOVA with Tukey's multiple comparisons test.
Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer (2021)
Figure 2

Figure 2. Combined inhibition of FGFR1 and PLK1 induces synergistic cytotoxicity in KRAS-mutant lung cancer and pancreatic cells

A, B, KRAS-mutant (n=17) and wild-type (n=14) cancer cell lines treated for 72h with AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination. The percentage of viable cells was color coded in a heatmap (A), with the combination index (CI) of AZD4547/BI2536 across the cell lines with different driver mutations shown in (B). CI < 0.8 indicates strong synergistic effect.

C, Clongenic assay of KRAS-mutant lung and pancreatic cells (MIA PaCa-2, H2122, H2009, H23, H358 and A549) treated with control (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination.

D, Apoptotic assay of KRAS-mutant cancer cells (H358, H441, A549, and SW620) and KRAS-WT lung cancer cells (EBC-1) treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 48h. Data are presented as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.

E, KRAS mutant lung (A549, H358，H441), pancreatic (MIAPaCa-2) and colon (SW620) cancer cells were transfected with control siRNAs, FGFR1- and PLK1-specific siRNAs, alone or combination. Cell viability was determined 72 h post transfection. Data are presented as mean ± SD (n=3). **p<0.01, ****p<0.0001, and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. ns, no significance.

F, Growth curve of a KRAS-mutant patient-derived xenograft (BE564T) treated with vehicle, AZD4547 (10 mg/kg/per day), BI6727 (5 mg/kg/per day), alone or in combination. *P<0.05 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD

G, H, Relative tumor volume (G) and weights (H) of the PDX (BE564T) after the treatment. *P<0.05 by unpaired two-tailed t-test. (H) Data are the mean of tumor weight of each group (5 mice/group); error bar: SD
Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer (2021)
Figure 4

Figure 4. ROS-activated JNK/p38/E2F1 axis contributes to the synergy of FGFR and PLK1 inhibitors

A, Immunoblots of H358 and A549 cells treated with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h.

B, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.

D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.

F, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.
Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer (2021)
Figure 5

Figure 5. Autophagy protects from the cytotoxicity of FGFR1/PLK1 inhibitors

A, Immunoblots of H358 and A549 cells treated for 24h with vehicle (DMSO), AZD 4547 (5 µM) and BI2536(5 nM), alone or in combination.

B, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm.

C, A549 and H358 cells expressing mCHerry-GFP-LC3B were treated with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h before flow cytometry-based analysis for mCherry and GFP (upper). Shown underneath is quantification of the data. **P<0.01 and ***P<0.001 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3).

D, Apoptotic assay of A549 and H358 cells treated with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5 nM) for 48h in the presence or absence of HCQ (10 µM). Data are presented as mean ± SD (n=3). *P<0.05 and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test.

E, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.

F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM).
Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer (2021)
Figure 6

Figure 6. A triple combination therapy for KRAS-mutant lung cancer

A, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD

B, C, Relative tumor volume (B) and weights (C) of PDX (BE564T) tumors after the treatment. *P<0.05 and ***P<0.001 by unpaired two-tailed t-test. (C) Data are the mean of tumor weights of each group (5 mice/group); error bar: SD

D, E, H&E and IHC analysis (Ki67 and Caspase-3) of PDX (BE 564T) tumors after the treatment (D). Quantification of the IHC data for the positivity of Ki-67 and Caspase-3 are shown in (E). *P<0.05 and **P<0.01 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3).

F-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group).
Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer (2021)
Figure 7

Figure 7. In vivo efficacy of the combination therapy in Kras-induced lung adenocarcinoma

A, Schematic of GEMM of Kras-induced adenocarcinoma. Established lung tumors in LSL-KrasG12D mice were treated with vehicle, AZD4547 (10 mg/kg/day), BI6727 (5 mg/kg/day), and HCQ (30 mg/kg/day) as indicated.

B, Lung morphology and H&E staining of lung tissue sections after 3-week treatment.

C, Tumor burden, average tumor size and tumor number (determined by QuPath software) after the treatment. *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. Data are presented as mean ± SD (error bar) of three or four replicates (n=3 or 4).

D, The tumor size in different treatment groups, calculated by number of tumors at the indicated size range divided by total tumor number of the treatment group. Data are the mean of four replicates (n=4).

E, Tumor grade analysis (% of nodules/lung) after the treatment. The anaylsis was based on DuPage et al, (2009). Data are the mean of three replicates (n=3).

F, Box plots showing the percentage of Ki67-positive cells in different treatment groups. Data are based on IHC staining of lung tissue section of LSL-KrasG12D mice. *P<0.05 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. The boxplots span from the first to third quartile, the whiskers extend to 1.5× the interquartile range (IQR). The horzontal line within the boxes represents the median of each group of 10 replicates (n=10).

G, Working model depicting synthetic lethal interaction of FGFR1/PLK1 inhibitors and autophagy as a protective mechanism in KRAS-mutant cancer cells.
Early Disruption of Photoreceptor Cell Architecture and Loss of Vision in a Humanized Pig Model of Usher Syndrome (2022)
Figure 4

Figure 4. Lack of harmonin does not alter expression of other USH proteins.

(A) RT-PCR was used to detect any USH1C transcripts (green arrows) and for discriminating USH1C_a and _c splice variants (blue arrows) from USH1C_b3 (orange arrows).

(B) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs, except in the heart and cerebellum. RT-PCR for GAPDH was used as control (Ctrl.).

(C) USH1C_a or _c splice variants were detected in the retina, kidney and intestine (blue arrows). Electropherogram from the duodenum, confirm alternative splicing of exon 13, but not exon 11. USH1C_b variants were also detected in all examined organs, with presumably two splice variants (orange arrows). Electropherogram from the colon, indicates alternative splicing of exon 20 as presumable reason for the double band. Exon 27 was not detected in any amplicon of USH1C_b RT-PCR (Figure S10D, E). Sequencing was done with a forward primer.

(D) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks (3w) (1 piglet, 1 retina, 1 duodenum) and 1 year (1y) (2 pigs, 1 retina each, 3 TRs). Arrow indicates the expected size of harmonin isoform a (72kDa).

(E) Representative immunofluorescence staining of harmonin (green) of longitudinal cryosection through WT and USH1C pig retinas (2 pigs, 1y, 1 retina each, 3 TRs, scale bar 10µm) demonstrated the absence of harmonin staining in the outer segment (OS) of photoreceptor cells and in the other layers below the outer limiting membrane (OLM, arrow) of the USH1C pig retina.

(F) Representative Western blot analysis of retinal protein extracts, indicating that the abundance of the USH proteins SANS (USH1G), whirlin (USH2D) or myosin7a (USH1B) is unaffected (1 piglet, 3w, 1 retina each, 2 TRs).
Early Disruption of Photoreceptor Cell Architecture and Loss of Vision in a Humanized Pig Model of Usher Syndrome (2022)
Figure 5

Figure 5. USH1R31X induce upregulation of GFAP and a synaptic phenotype in USH1C pig photoreceptor cells.

(A, B) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks (3w). (A) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM (arrow) to the ganglion cell layer (GCL) of the retina. The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; scale bar: 20 µm. (B) Left panel: Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls. Anti-actin Western blot was used as loading control. Right panel: Quantification of Western blot bands in 4 gels by the LI-COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age-matched WT controls (2 piglets, 3w, 2 retinas each, 2 TRs, error bars represent standard deviation (SD) of the mean, two-tailed Student's t tests, *p<0.05).

(C, D) Reduced synaptic width in USH1C pigs at an age of 1 year (1y). (C) Fluorescent microscopic analysis of the cone synapse phenotype. Left panel: Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre-synaptic marker PSD-95 (green) and by fluorescent peanut agglutinin (PNA, red) for cone synaptic pedicles (white arrows) and counter-stained by DAPI for nuclear DNA (blue). Scale bar 10µm. Middle panel: Higher magnification of a PNA-stained cone synaptic pedicle. Synapse width was determined as the maximum extension of consistent PNA signals. Scale bar 1µm. Right panel: Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA-stained sections indicated reduced synaptic width (2 pigs, 1y, 1 retina each, number of examined synapses indicated, error bars represent SD, two-tailed Student's t tests, ***p<0.001). (D) Determining cone synaptic pedicles width by TEM. Left panel: Representative images of retinal cross sections. Scale bar 500nm. Right panel: Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs. (1 pig, 1y, 1 retina each, number of examined synapses indicated, error bars represent SD, two-tailed Student's t tests, *p<0.05).
Early Disruption of Photoreceptor Cell Architecture and Loss of Vision in a Humanized Pig Model of Usher Syndrome (2022)
Figure 6

Figure 6. Changes in ciliary structures and outer segment architecture of USH1C photoreceptor cells.

(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).

(D, E) Measuring CC length in TEM sections. (D) Representative images of longitudinal cross sections and principle of determining CC length. Scale bar 500nm. (E) Elongation of CC length is confirmed in TEM sections of USH1C PRC. (1 pig, 1y, 1 retina each, number of examined connecting cilia indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).

(F-H) Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (G) Left panel: The number of ciliated cells is decreased in porcine fibroblasts USH1C pigs, compared to WT controls (6 TR, number of examined cells indicated, error bars represent SD, two-tailed Student's t tests, *p<0.05). Right panel: The length of primary cilia in porcine fibroblasts is increased in USH1C (6 TR, number of cells examined indicated, Kolmogorov-Smirnov (KS)-test, ****p<0.0001). (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001).

(I-M) Outer segment (OS) PRC architecture by TEM of longitudinal retinal sections. (I) At 3w, WT show normal sub-cellular organization, including parallel stacking of photosensitive discs and calyceal processes (black arrows, representative image from 2 pigs, 2 retinas, scale bar 600nm). (J) In 3w USH1C rods, membrane discs appear also in vertical orientation (white arrows, representative image from 2 pigs, 2 retinas, scale bar 750nm). At 1y, interstitial gaps appeared in disc stacks of the OS in USH1C pigs (K, arrow, scale bar 550nm) and vesicles are found at the OS base (L, asterisk, scale bar 500nm). (M) The disc architecture in 1y animals was substantially distorted (arrows, representative image from 2 pigs, 2 retinas, scale bar 850nm).

(N) Localization of cellular anomalies in an USH1C rod highlighted in orange.
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 1

Figure 1. AtANN1 mediates plants response to freezing tolerance in CBF-dependent manner.

A-C Freezing phenotype (A), survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Representative pictures are shown in (A).

D,E Expression of CBFs (D) and their targets (E) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (D) or 24 h (E), and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.

Data information: In (B and C), the data represent means ± SE of three independent experiments, each with three technical repeats (n=25 seedlings for each repeat). **P < 0.01, two-tailed t-test. In (D and E), the data represent means ± SD of three technical replicates (**P < 0.01, * P < 0.05, two-tailed t-test). Three independent experiments showed a similar result.