EMBO/soda-nel huggingface dataset:
Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication  (2020)
Figure 3

Figure 3. Translocation of the deletion variants of CNFY and their influence on RhoA activation, actin rearrangements and multi­nucleation of host cells.

A HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants fused to β-lactamase (TEM) at 37°C for 4 h. Cleavage of the reporter dye CCF4-AM was used to visualize toxin delivery. After cell entry CCF4-AM is rapidly converted into the negatively charged form CCF4, which is retained in the cytosol and emits a green fluorescence signal (520 nm). In the presence of trans­located β-lactamase fusion proteins, CCF4-AM is cleaved, and disruption of FRET results in blue fluorescence (447 nm). Scale bar: 20 µm.

B Left upper and right panel: HEp-2 cells re­mained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 4 h. Cells were lysed and the de­amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; left lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNFY or the N-ter­minally deleted toxin variants for 4 h. The de­amidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE after detection with anti-RhoA antibodies.

C HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was ob­served by fluorescence micro­­scopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY-treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.
Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication  (2020)
Figure 4

Figure 4. Localization of the N- and C-terminal deletion variants of CNFY in the late endosome.

HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY, N- or C-terminal deletion variants fused to GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and smaller images are magnified views of boxed areas. White scale bar is 10 µm.
Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication  (2020)
Figure 6

Figure 6. Characterization of mutants in the linker region connecting domain D3 and D4.

A HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing CNFY, the toxin variant mut1: CNFYI535L/P536A/V537G or mut2: CNFYI535L/P536A/­V537G/F539L/­D541A/K542A fused to TEM or no CNFY protein (empty vector) for 4 h. Cells were lysed and the binding of the different CNFY proteins to HEp-2 cells was analyzed by immunoblotting.

B HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing CNFY, the toxin variant mut1: CNFYI535L/P536A/V537G or mut2: CNFYI535L/P536A/­V537G/F539L/­D541A/K542A or no CNFY protein (empty vector). The cell nuclei were stained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The results indicated the formation of polynucleated cells and stress fibers only in cells treated with CNFY and CNFYI535L/­P536/V537G. The white scale bar is 20 µm.

C Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β-lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.

D The viability of Y. pseudotuberculosis YPIII expressing the indicated CNFY derivatives was assessed in equalized bacterial cultures using the BacTiter-Glo Microbial Cell Viability Assay kit (Promega). The data represent the mean ± SD of three independent experiments, carried out in triplicates.

E HEp-2 cells treated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing indicated CNFY variants for 4 h were lysed and the deamidation of RhoA was analyzed by the mobility shift of the modified RhoA GTPase detected by immunoblotting.

F The activity of the CNFY derivatives was tested by analyzing the deamidation of RhoA in HEp-2 cell lysates by the mobility shift of the modified GTPase detected by immunoblotting.
Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication  (2020)
Figure 7

Figure 7. Structure and deamidation activity of the free D4-5 subunit of CNFY.

A Crystal structure of the free D4-5 subunit. Note the different relative orientations of domains D4 and D5 with respect to the structure of full-length CNFY (top, thin grey lines). The domain D4 forms a large interface area (1100 Å2) with the catalytic domain D5 involving several polar interactions (8 hydrogen bonds and 8 salt bridges), whereby the active crevice is extended and fully solvent-exposed as can be seen in the right surface plot at the bottom of the panel. The hypothetical NAD+ binding site of the ART-like D4 domain is located on the opposite face (left surface plot). Note that the deamidase active site of domain D5, unlike in the full-length structure (Fig 1), is fully accessible and that its extended shape is also determined by domain D4.

B Comparative analysis of RhoA activation in HEp-2 cell lysate by CNFY and the recombinant D4-5 protein. Purified CNFY or the D4-5 fragment (1 µM) was added to extracts of HEp-2 cells and incubated for 10, 20, 30 or 60 min. De­amidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels after detection with anti-RhoA antibodies.

C Comparative analysis of recombinant RhoA deamidation by CNFY and the D4-5 fragment. Recombinant RhoA was incubated with purified CNFY or the D4-5 fragment and samples were separated by SDS-PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry. Error bars represent standard deviations of triplicate measurements.
Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies (2021)
Figure 1

Figure 1. CAF-1 promotes HIV-1 latency.

A RNA-Seq of naïve and TNFα-stimulated J-Lat 10.6 cells. Differentially expressed genes were filtered with log2FC of 1 and PvalueFDR cutoff of 0.05. Upregulated and downregulated genes were labeled as red and blue dots respectively. Representative genes were labeled aside corresponding dots.

B RNA-Seq result as in (A). Significantly changed genes were sorted out and plotted as heatmap. SEC16A indicated unchanged gene.

C The GFP-positive percentages of monoclonal sgCHAF1A and sgNT J-Lat 10.6 cell lines were shown in the top right corner of each flow cytometry figure. TNFα, SAHA and JQ-1 were used as supplements.

D ChIP assay with antibody against CHAF1A was performed in J-Lat 10.6 cells. All the ChIP-qPCR DNA signals were normalized to siNC IgG of G5'. G5' represented cellular DNA and viral 5'LTR junction; A: Nucleosome 0 assembly site; B: Nucleosome free region; C: Nucleosome 1 assembly site; V5: Viral 5'LTR and gag leader sequence junction; E represented envelope; V3: Viral poly purine tract and 3'LTR junction; G3' represented viral 3'LTR and cellular DNA junction.

E The statistical graph of the result in (C).

F-I ChIP assays with antibodies against H3K9me3, H4K20me3, H3K4me3 and H3K9Acetyl were performed as in (D). Only 'C' position signals were showed and normalized to Input.

J, K J-Lat 8.4 cells were treated with shCHAF1A lentiviruses and 5-aza-dC. The GFP-positive cells and HIV-1 LTR methyl-CpGs percentages of different groups were plotted in (J) and (K) respectively.

L ATAC-Seq was performed in WT and CHAF1A-KO J-Lat 10.6 cells. The relative tag densities of the pseudotyped HIV-1 5'LTR integration site in each group were calculated. The highest tag density was set as 100. Figure showed 2kb range centered the 5'LTR integration site. Dashed line represented HIV-1 pseudotyped virus 5'LTR integration site.

M ChIP assays with antibodies against CDK9 and Pho-Pol II were performed in siNC and siCHAF1A J-Lat 10.6 cells. ChIP signals in each group were normalized to Input.

Data information: Data represented mean ± SEM in triplicate. p-Values were calculated by Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies (2021)
Figure 3

Figure 3. CAF-1 forms nuclear bodies.

A Super-resolution SIM image of CHAF1A in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibody was used to label endogenous CHAF1A which was colored into green.

B Super-resolution cSTORM image of CHAF1A in HEK293T cells. Two degrees of amplification were performed to show CAF-1 bodies.

C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα.

E RFP-tagged CHAF1A and GFP-tagged PML body component SP100 were co-overexpressed in HEK293T cells and imaged by SIM.

F GFP-tagged CHAF1A and RFP-tagged BRD4 were co-overexpressed in HEK293T cells and imaged by SIM.

G-M SUV39H1, HP1α, DNMT1, MBD1, RING1B, EZH2 and SUMO4 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination.

N-P GFP-tagged CHAF1A was overexpressed in HEK293T cells. CF568-tagged antibodies against H3K9me3, H3K27me3 and H3K4me3 were used to treat IF samples respectively. CHAF1A and each histone modification were imaged by SIM.

Data information: The scale bar of each SIM image represented 5 μm. The scale bars of amplified STORM images in (B) represented 1 μm (primary amplification) and 0.1 μm (secondary amplification). All the samples were imaged to obtain at least three images.
Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies (2021)
Figure 4

Figure 4. CAF-1 bodies are phase-separated nuclear condensates.

A FRAP images of CAF-1 bodies to indicate the internal diffusion property. GFP-tagged CHAF1A was overexpressed in HEK293T cells. Twenty-four hours later, live cells were proceeded to time series imaging. One of the CAF-1 bodies was bleached with intense laser pulse. Images of live cell were captured every 4 seconds. One of the unbleached CAF-1 bodies was used as control. Data represented eight time points. At least three cells of each sample were treated with intense laser pulse to obtain bleached CAF-1 bodies.

B Representative images of two CAF-1 bodies fusing into one.

C Representative images of one CAF-1 body splitting into two.

D Live cells were imaged every 2 seconds in time series. 1,6-hexanediol was added into the cell incubation well carefully at time point of 5th second. Images were captured for 3 minutes.

E GFP-tagged CHAF1A-N-NLS and CHAF1A-C were imaged with RFP-tagged CHAF1A respectively. N: CHAF1A N-terminal. NLS: nuclear localization signal of CHAF1A, which was presented within C-terminal. C: CHAF1A C-terminal.

F Upper panel showed WT-CHAF1A bodies. Lower panel showed dLLPS-CHAF1A which was CHAF1A-Q34A-I116A-K395D-R402D-K409D.

G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.

Data information: The scale bar of images in (A-F) represented 5 μm. The scale bar in (G-I) represented 10 μm.
Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies (2021)
Figure 5

Figure 5. CHAF1A is the leading factor of CAF-1 body.

A The distribution of CHAF1B in the presence of CHAF1A (upper panel) and in the absence of endogenous CHAF1A (lower panel).

B First panel: the co-localization of PCNA and CHAF1A. Second panel: the distribution of PCNA in the absence of endogenous CHAF1A. Third panel: the distribution of PCNA in the presence of CHAF1A-dPIP1 mutant and the absence of endogenous CHAF1A. Fourth panel: the co-localization of PCNA and over-expressed DNMT1 in the absence of endogenous CHAF1A.

C Upper two panels: the distribution of HP1α in the presence or absence of CHAF1A. Lower two panels: the distribution of HP1α in the presence of CHAF1A-dHP1BD or CHAF1A-DS2-dHP1BD. Both images of lower two panels were in the absence of endogenous CHAF1A.

D-F The distribution of SUV39H1 (D), SUV39H2 (E) and MBD1 (F) in the presence or absence of CHAF1A.

G The distribution of SUMO paralogs in the absence of CHAF1A.

Data information: The scale bar within each image represented 5 μm.
Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies (2021)
Figure 6

Figure 6. LLPS-deficient CAF-1 body is unable to mediate HIV-1 latency.

A Flow cytometry figures of CAF-1 body rescue experiment. First figure: Wildtype J-Lat 10.6. Second figure: CHAF1A knock-out J-Lat 10.6. Third figure: CHAF1A-KO J-Lat 10.6 was re-introduced with wildtype CHAF1A. Fourth figure: CHAF1A-KO J-Lat 10,6 was re-introduced with dLLPS-CHAF1A which harbored five mutations. The percentages of GFP-positive cells were labeled in the top right corner of each figure.

B The statistical histogram of the results in (A).

C Rescue experiment in heterogeneous Jurkat latency model which were performed as in (A).

D, E The enrichment of H3K9me3 and H3K27me3 on HIV-1 LTR in different CHAF1A status. The reactivation efficiency in each group was indicated by the percentage of GFP-positive cells.

Data information: Data represented mean ± SEM in triplicate. p-Values were calculated by Student's t-test. **p < 0.01, ***p < 0.001.
Histone chaperone CAF-1 promotes HIV-1 latency by leading the formation of phase-separated suppressive nuclear bodies (2021)
Figure 7

Figure 7. CHAF1A depletion reactivates latent HIV-1 from HIV-1-infected individuals.

A The procedure of CHAF1A depletion-mediated HIV-1 reactivation in primary CD4+ T cells. On Day -4, primary CD4+ T cells were isolated from PBMCs of healthy donors, and activated by PHA. On Day 0, activated CD4+ T cells were washed and infected with pseudotyped HIV-1. Another two days later, cells were treated with shluc and shCHAF1A lentiviruses respectively. Two weeks later, one part of naïve CD4+ T cells was treated with αCD3/αCD28/IL-2. One part of shluc-treated cells and one part of shCHAF1A-treated cells were further treated with 500 nM SAHA. On day 16, all the cells were proceeded to FCM analysis.

B The flow cytometry figures of each group. The reactivation efficiency was indicated by the GFP-positive cells percentages which were labelled on the top right corner.

C The statistical scatter plot of the results in (B).

D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement.

E Envelope V1 to V3 region of HIV-1 RNAs in (D) was reverse-transcribed and PCR-amplified. The PCR products were proceeded to TA-cloning. At least 60 single clones were picked from each group and sequenced. These sequences and the standard HIV-1 sequence HXB2 were aligned. Genetic diversity index of each sequence was calculated for each group.

F The schematic of CAF-1 body-mediated HIV-1 latency. Detailed information for the schematic was indicated in discussion.

Data information: Data in (C) represented mean ± SEM in sextuplicate. p-Values were calculated by Mann-Whitney U-test. Data in (D) represented mean ± SEM in triplicate. p-Values were calculated by Student's t-test. Data in (E) represented mean ± SEM (n=40 for each group within each patient sample). p-Values were calculated by Mann-Whitney U-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Expanded View Figure legends
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome (2021)
Figure 1

Figure 1: Overall abundance changes of mitochondrial proteins.

(A, B) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles (Dataset EV1). A, all mitochondrial proteins detected. Grey area indicates proteins that changed less than 2-fold in both directions. Subunits of selected mitochondrial machineries are highligted as follows: magenta, pyruate dehydrogenase complex (PDH); blue, mitochondial carriers (Carrier); brown, translocase of the outer membrane (TOM); yellow, translocase of the inner membrane (TIM); green, mitochondrial contact site and cristae organizing system (MICOS); yellow-green, mitochondrial fusion-fission system (Dynamics); red, porin or voltage dependent anion channel (VDAC); B, subunits of OXPHOS complexes (Complex I-V) and mitoribosomes (Ribosome). Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex. For the full gene name, add NDU (complex I), SDH (complex II), UQC (R10), COX (complex IV), ATP (5L, 5J2), MRP (Ribosome) in front of the abbreviated gene names given in the figure.
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome (2021)
Figure 3

Figure 3: Genetic system of mitochondria.

(A, B) Selected proteins involved in DNA (A) and RNA (B) maintenance of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, for POLG, UNG and TWNK, only abundances >1 MDa were quantified. Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test for individual proteins and two-way ANOVA for changes of multiple proteins in a complex).
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome (2021)
Figure 4

Figure 4: Energy metabolism of mitochondria.

(A-F) OXPHOS complexes (A-C) and selected enzymes involved in the energy metabolism of control (143B, solid lines) and ρ0 cells (dashed lines). A, Complex (C) I, III and IV; B, F1, FO domain and inhibitor protein IF1 of complex V; C, subunits SDHA, SDHB-D and assembly factor SDHAF2 of complex II; D, oxoglutarate (OGDH; average of OGDHL, OGDH, DLST) and pyruvate dehydrogenase complex (PDH; average of PDHA1, PDHB, PDHX, DLAT); E, subunits of GDP forming (SUCLG2) and ADP forming (SUCLA2) succinyl-CoA ligase; F, urea cycle enzymes and glutaminase (GLS). Plots and gene names are in corresponding colors.
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome (2021)
Figure 5

Figure 5: Mitochondrial transporters.

(A-D) Selected metabolite carriers of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, isoforms of the nucleotide carrier (ANT1-3) and phosphate carrier (PiC); B, pyruvate carrier; C, dicarboxylate carriers; D, mitoferrins (MFRN1-2) and glycine transporter (GlyC). Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01 (Student's t-test).
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome (2021)
Figure 6

Figure 6: Protein import machinery.

(A-D) Components of the mitochondrial protein import machinery and selected proteins related to this process of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored and dark-grey bars). A and B, components of the translocase of the outer membrane (TOM) machinery (TOM avg.: average of TOMM40, TOMM22, TOMM5, TOMM6, TOMM7); C, components of the trans­locase of the inner membrane (TIM) machinery (TIM22 avg.: TIMM22, AGK, TIMM10B; TIM23 avg.: TIMM23, TIMM17A, TIMM17B); D, components of the TIM23-associated TIMM21 assembly machinery (left panel) and its association with prohibitin (PHB) and components of the mitochondrial quality control system (right panel). Plots and gene names are in corresponding colors, except for TIMM44 and TIMM50 (no migration profiles shown). In (A-C) data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test).
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome (2021)
Figure 7

Figure 7: Mitochondrial morphology and dynamics.

(A-C) Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, mitochondrial contact site and cristae organizing system (MICOS: MIC60, MIC19, MIC25) and SAM complex (SAM: SAMM50, MTX2, MTX3) together forming the MIB complex; B, proteins involved in controlling mitochondrial morphology (orange) and dynamics (teal); C, DNM1L/DRP1 and interacting proteins. For DNM1L, MTFR1 and ARL2BP, quantification of the entire migra­tion profiles (Overall) and the range above 1,400 kDa only (>1,400 kDa) are shown. Plots and gene names are in corresponding colors, except for ARL2 (dark-grey) not shown in the plot. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).
LIN28A loss-of-function is associated with Parkinson's disease pathogenesis (2019)
Figure 1

Fig. 1. Developmental defects associated with the pathogenesis of Parkinson's disease (PD) in Nestin-Cre/Lin28a/b conditional knockout (cKO) mice. (A-C) Defects in midbrain-type dopamine (mDA) neuron-specific developmental factor expressions in the ventral midbrain (VM) of Lin28a/b cKO embryos. Embryonic VM sections from 3 WT and cKO animals each were immunoflorescent stained for the midbrain-specific markers in parallel and mean fluoresecent intensities (MFI) were measured. n = 20 cells for each group. *** p<0.0001, t-test. (D, E) Reduced mDA neurons in the embryonic VM (E12.5) and adult SN at 2-7 months (young age) and at 14-18 months (old age) of the Lin28a/b cKO mice. *p<0.05, **p<0.005, n=4 each wild-type (WT) and cKO embryos, and 9 (WT), 8 (cKO) in adult mice. (F, G) Enhanced DA transporter (DAT)+ and TH+ fiber intensities of Lin28a/b cKO adult mice (age: 4-7 months). Striatal sections of WT and cKO mice (3 animals for each group) were subjected to immunofluorescent staining against DAT and TH in parallel, and MFI was measured from 101 microscopic fields of dorsolateral striatum. (H) DA uptakes assessed by nanoScanPET/MRI analysis using [18F]FP-CIT. n=9 (WT), 8 (cKO). *p<0.05, ***p<0.001. (I-M) Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. WT and cKO mice were injected with MPTP (20mg/kg, i.p) for 5 days and behaviors of the animals were assessed for 1 month after the MPTP injection (I-K). The mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH+ mDA neuron counts at SN (L) and DAT MFI at striatum (M). n= 8-13 (WT), 5-9 (cKO). *p<0.05, **p<0.01, ***p<0.0001.