EMBO/soda-nel huggingface dataset:
The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses (2022)
Figure 2

Figure 2. DDX42 is an intrinsic inhibitor of HIV-1.

A. Top: DDX42 KO and CTRL KO U87-MG/CD4/CXCR4/Cas9/Firefly cells were generated using 3 sgRNAs and 4 non-targeting sgRNAs, respectively (for CTRL, the average of data obtained with 4 cell populations is shown). Cells were treated or not with IFN 24 h prior to infection with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Relative luminescence results for IFN-treated and -untreated conditions are shown. Two-way ANOVA on log-transformed data with Sidak's test. Bottom: Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42-depleted populations; Actin served as a loading control.

B. siRNA-transfected U87-MG/CD4/CXCR4 cells were treated or not with IFN for 24 h prior to infection with HIV-1 Renilla. Relative luminescence results for IFN-treated and -untreated conditions are shown. Multiple linear regression analysis.

C. DDX42 silencing efficiency measured by RT-qPCR (top) and immunoblot (bottom) in parallel samples from B.

D. DDX42-depleted cells were infected with HIV-1 (WT NL4-3), and infection efficiency was measured by CA p24Gag intracellular staining and flow cytometry analysis. When indicated, cells were treated with azidothymidine (AZT) and lamivudine (3TC). Two-way ANOVA on log-transformed data with Dunnett's test.

E. Ctrl and IRF9/STAT1 KO cells were pre-treated or not with IFN for 24 h and infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Infection efficiency was assessed 24 h later by measuring Renilla activity. Two-way ANOVA on log-transformed data with Dunnett's test.

F. siRNA-transfected MDMs were infected with a CCR5-tropic version of HIV-1 Renilla (NL4-3/R5/Nef-IRES-Renilla). Relative luminescence results from biological triplicates performed with cells from different donors are shown. Two-way ANOVA on log-transformed data with Dunnett's test.

G. Primary CD4+ T cells were electroporated with Cas9-sgRNA RNPs using 2 non-targeting sgRNAs (sgCTRL1 and 2) and 5 sgRNAs targeting DDX42. Top: Cells were then infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and relative infection efficiencies obtained with cells from 3 donors are shown. Two-way ANOVA on log-transformed data with Dunnett's test. Bottom: DDX42 protein levels were determined by immunoblot, Actin served as a loading control. A representative immunoblot is shown.

H. Firefly- or DDX42-expresssing U87-MG/CD4/CXCR4 cells were infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Relative infection efficiencies are shown. Multiple linear regression analysis.

Data information: A-D and F-H, Data represent the mean ± S.E.M of 3 biological replicates, E, Data represent the mean ± S.E.M of 4 biological replicates. P values are denoted as follow: ns not significant, *p<0.05, **p<0.01, ****p<0.0001.
The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses (2022)
Figure 5

Figure 5. DDX42 is found in close proximity to viral components and interacts with viral RNAs.

A-B. (A) Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA using mouse anti-dsRNA (J2) and rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition). Mann-Whitney test. (B) Identical to A but using an anti-N antibody instead of anti-dsRNA antibody.

C-E. (C) Left, Quantification of SARS-CoV-2 RNA by RT-qPCR in RNA from total U87-MG-ACE2 cell lysates (input) and in Flag-Firefly (negative control) and Flag-DDX42 immunoprecipitation (IP). Multiple linear regression analysis on log-transformed data. Right, Percentage of immunoprecipitated RNA. Paired t-test on log-transformed data. (D) Identical to C following CHIKV infection of U87-MG cells. (E) Identical to C following IAV infection of U87-MG cells.

F. The following biotinylated RNAs were used to pull-down recombinant DDX42 (Fig. EV3B): poly(I:C), CHIKV G4, TRF2 G4 and a mutated TRF2 G4 sequence (Mut. TRF2 G4), and DDX42 was revealed by an immunoblot. A representative experiment out of 2 biological replicates is shown.

Data information: A-E Data represent the mean ± S.E.M. of 3 biological replicates. P values are denoted as follow: ns not significant, *p<0.05, ****p<0.0001

Extended View Figure legends
Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway (2016)
Figure 1

Figure 1. Mcp3 is a multi-copy suppressor of mdm10∆. (A) Rescue of growth phenotype. Wild-type (WT) or mdm10∆ cells were transformed with the empty plasmid pYX142 (). In addition, mdm10∆ cells were transformed with pYX142 encoding MDM10 or MCP3. Cells were grown to an OD600 of 1.0 and spotted on YPD or YPG plates in a 1:5 dilution series. Plates were incubated for growth at the indicated temperatures. (B) Over-expression of Mcp3 partially rescues the mitochondrial morphology defect in mdm10∆ cells. Cells described in (A) expressing mitochondrially targeted GFP (mtGFP) were analysed by fluorescence microscopy. Typical images (fluorescence and brightfield, BF) of the four different strains are shown (scale bar = 5 µm). The quantification shows the average percentage with standard deviation bars of three independent experiments (n=3; SD; *, p < 0.05 and as indicated) with at least 100 cells per experiment.
Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway (2016)
Figure 2

Figure 2. Mcp3 rescues assembly defects of protein complexes and lipid alterations of mdm10∆ cells and restores growth of other ERMES deletion mutants. (A) Respiratory chain super-complex levels are slightly restored by over-expression of Mcp3. Mitochondria isolated from the indicated strains were lysed in 1% digitonin and subjected to a 4-8% BN-PAGE. Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV. (B) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over-expression. Mitochondria of the indicated cells were lysed in 1% digitonin and subjected to a 6-13% BN-PAGE and immunoblotting with antibodies against Tom40 (left) or Tob55 (right). The assembled TOM complex and an unassembled Tom40 species at ca. 100 kDa (arrowhead) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex (asterisk). (C) Over-expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10. Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometric analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from other cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD; *, p < 0.05) is given. (D) Mcp3 rescues loss of Mdm12. Wild-type (WT) or mdm12∆ cells transformed with the empty plasmid pYX142 () or mdm12∆ cells over-expressing either Mcp3 or Mdm12 (as a control) were analysed at 30°C or 37°C by drop dilution assay on rich medium containing glycerol (YPG). (E) Mcp3 rescues loss of Mmm1. WT and mmm1∆ cells were transformed and analysed as described in (D). (F) Mcp3 rescues loss of Mdm34. WT and mdm34∆ cells were transformed and analysed as described in (D).
Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway (2016)
Figure 5

Figure 5. Mcp3 is imported by the TOM complex. (A) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3. p and m, precursor and mature forms, respectively. (B) Import of Mcp3 is dependent on the receptor Tom70. Mitochondria isolated from wild-type or tom70/71∆ cells were incubated with radiolabelled Mcp3 or pSu9-DHFR (as control) for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. p and m, precursor and mature forms, respectively. I, 20% of radiolabelled precursor protein used in each import reaction. Bands corresponding to the mature (m) form were quantified. Import into wild-type mitochondria after 15 min was set to 100%. The mean with standard deviations is depicted (n=3; SD). (C) Reduced steady state levels of HA-Mcp3 in cells lacking Tom70. Crude mitochondria isolated from the indicated cells were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, AAC as control substrate of Tom70, and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05). (D) Import of Mcp3 is dependent on Tom40. Mitochondria isolated from wild-type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods. Next, mitochondria were reisolated and analysed by SDS-PAGE and autoradiography as described in (B). As a control Ugo1 was imported into the same mitochondria. Organelles were reisolated and analysed by BN-PAGE and autoradiography. The band corresponding to the Ugo1 dimer at around 150 kDa was quantified. The mean with standard deviation is shown (n=3; SD). (E) Steady state levels of HA-Mcp3 are lower in cells harbouring a mutant TOM40 allele. Crude mitochondria from wild-type and tom40-25 cells were obtained and analysed as described in (C). Immunodecoration was performed with antibodies against the HA-tag, Tom40, and Fis1 as loading control.
Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway (2016)
Figure 6

Figure 6. Mcp3 precursor physically interacts with components of the TOM complex. (A) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing bound material was eluted with sample buffer. Proteins were analysed by SDS-PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used. (B) Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His-Tom22 containing cells. Mitochondria were pre-incubated with CCCP (40 µM) to halt import. After lysis with β-dodecyl maltoside samples were incubated with Ni-NTA beads. After washing bound proteins were eluted with sample buffer and analysed by SDS-PAGE and autoradiography. I, 5% of 35S-Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni-NTA; n.b. 2% of non-bound material after binding to Ni-NTA; eluate, 100% of bound material. (C) Radiolabelled Mcp3 was incubated with organelles described in (B). After import mitochondria were solubilized in digitonin, the lysate was cleared by centrifugation and incubated with or without an antibody against the His-tag (α-His). Samples were analysed by BN-PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti-His antibody are indicated by arrowhead.
Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway (2016)
Figure 8

Figure 8. Insertion of Mcp3 into the MOM involves the MIM complex. (A) Import of Mcp3 is mediated by the MIM complex. Mitochondria isolated from WT, mim1∆, or mim2∆ cells were incubated with radiolabelled Mcp3 for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. Bands corresponding to the mature (m) form were quantified. Import after 15 min into wild-type mitochondria was set to 100%. The mean with standard deviations is depicted (n=3; SD). I, 20% of radiolabelled precursor protein used in each import reaction (B) Steady state levels of over-expressed HA-Mcp3 are reduced in cells lacking MIM subunits. Crude mitochondria were obtained from WT cells containing an empty plasmid (Ø) and from wild-type, mim1∆ or mim2∆ cells containing a plasmid encoded HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, Tom20 as MIM substrate and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in wild-type cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05; **, p < 0.01). (C) Mcp3 precursor binds in vitro to Mim1. Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1. After washing proteins were eluted with sample buffer. Analysis was performed by SDS-PAGE, blotting and autoradiography. To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used; n.b., 5% of unbound material;.b, 100% of bound material.
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 1

Figure 1. Mouse osteoclasts express Zeb1 in vitro and in vivo.

A DAVID gene ontology (GO) analysis of differentially upregulated transcripts during BMDM-osteoclast transition generated from wild-type mice identified an enrichment in DNA-binding TF activity (arrow; n = 3).

B Unexpected expression of Zeb1 (arrow) among the top 20 differentially upregulated genes retrieved from DAVID GO biological process "DNA-binding TF activity" during wild-type BMDM-osteoclast transition (n = 3).

C Relative mRNA expression of Zeb1, Acp5, and Ctsk during the differentiation from BMDMs to osteoclasts in culture (n = 3).

D Western blot of Zeb1, c-Src, and Ctsk expression in BMDMs and mature osteoclasts (n = 3).

E 3D reconstruction of Zeb1 (red), TRAP (green), and F-actin (cyan) immunofluorescent staining of osteoclasts differentiated from wild-type BMDMs. Scale bar, 20 μm.

F Zeb1 (red) and TRAP (green) immunofluorescence of a femur section including the growth plate (GP) and metaphysis from a wild-type mouse. The multinucleated cells associated with the surface of the bone marrow cavity (BM cavity) that are shown in the red box are further enlarged in the panels to the right. Scale bar, 10 μm.

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using unpaired Student's t test (C). **P < 0.01.

Source data are available online for this figure.
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 2

Figure 2. Zeb1 conditional knockout mice are osteopenic with heightened bone resorptive activity in vivo.

A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown. The genotype of Zeb1+/+, Zeb1f/+, Zebf/f mice is defined as "wild-type" control. Scale bar, 500 μm.

B Quantification of BV/TV, Tb.Th, Tb.N, and Tb.Sp as determined by nanoCT in 3-month-old wild-type and Zeb1ΔM/ΔM male mice (n = 8).

C H&E and TRAP staining of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice. Scale bar, 200 μm.

D Quantification of osteoclast number per bone surface (N.Oc/BS) and eroded surface per bone surface (ES/BS), as well as serum CTX-I and OCN levels in 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown (n = 6).

E Golden's trichrome staining illuminating howship's lacunae (arrow) of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice were performed along with double-calcein bone labeling and confocal imaging to assess bone formation. Scale bar, 10 μm.

F Quantification of MAR and BFR as assessed in 3-month-old male wild-type and Zeb1ΔM/ΔM mice (n = 6).

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using unpaired Student's t test (B, D, F). ns, not significant; **P < 0.01.
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 3

Figure 3. Enhanced sealing zone formation and bone resorbing activity of Zeb1-knockout osteoclasts in vitro.

A BMDMs were isolated from wild-type or Zeb1ΔM/ΔM mice and cultured atop plastic substrata with M-CSF and RANKL for 5 d, stained with TRAP, and the number of TRAP+ MNCs determined (n = 6). Scale bar, 500 μm.

B Zeb1, NFATc1, c-Fos, c-Src, β3 integrin, Mmp9, Mmp14, and Ctsk expression as assessed by Western blot in wild-type and Zeb1ΔM/ΔM BMDMs during osteoclast differentiation (n = 3).

C, D After a 6-day culture period, wild-type or Zeb1ΔM/ΔM osteoclasts were removed from bone slices, resorption pits visualized by WGA-DAB staining (C), and resorption pit area quantified (D; n = 6). Scale bar, 100 μm.

E, F Resorption pits were 3D reconstructed by reflective confocal laser scanning microscope (RCLSM) (F) in tandem with quantification of resorption pit depth (E; n = 6). Color bar, pit depth.

G Supernatant CTX-I determined by ELISA (n = 6).

H, I After a 6-day culture atop bone slice, phalloidin staining (red) was performed in wild-type versus Zeb1ΔM/ΔM osteoclasts (I), and actin ring area per cell quantified (H). Scale bar, 20 μm.

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using unpaired Student's t test (A, D, E, G, H). ns, not significant; *P < 0.05, **P < 0.01.

Source data are available online for this figure.
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 4

Figure 4. Zeb1 represses MtCK1 expression and tunes the phosphocreatine energy shuttle in osteoclasts.

A Volcano plot showing significantly enriched and depleted mRNAs in Zeb1ΔM/ΔM osteoclasts versus wild-type osteoclasts (n = 3).

B The top 10 most highly upregulated and downregulated transcripts in Zeb1ΔM/ΔM osteoclasts as compared with wild-type osteoclasts are presented (n = 3; color bar, relative expression value). C DAVID gene ontology (GO) analysis of differentially expressed genes from Zeb1-deleted versus wild-type osteoclasts.

D Mitochondrial creatine kinase activity of wild-type and Zeb1ΔM/ΔM BMDMs and osteoclasts cultured atop plastic substrata (n = 3).

E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice.

F MtCK1, Ckb, VDAC, and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).

G MtCK1 and Ckb expression in the mitochondrial fraction as assessed by Western blot in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).

H MtCK1 (green) immunofluorescence of wild-type and Zeb1ΔM/ΔM osteoclasts. Scale bar, 20 μm.

I Schematic of mouse Ckmt1 promoter region indicating locations of E-box (red) and Z-box (blue) sequences that represent putative Zeb1 binding sites.

J Luciferase activity was monitored in RAW 264.7 cells co-transfected with a mock or ZEB1 expression vector in combination with a Ckmt1 promoter reporter construct (n = 3).

K qPCR analysis of ChIP using anti-Zeb1 antibody at the Ckmt1 promoter region in mature osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using two-way ANOVA with Bonferroni correction (D, E, J, K). ns, not significant; *P < 0.05, **P < 0.01.

Source data are available online for this figure.
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 5

Figure 5. Zeb1 depletion increases mitochondrial respiration and ATP production in osteoclasts.

A Phosphocreatine (PCr) and creatine (Cr) level, as well as PCr/Cr ratio of wild-type or Zeb1ΔM/ΔM osteoclasts cultured atop plastic substrata as determined by LC/MS analysis (n = 3).

B, C Oxygen consumption rate (OCR) profile plot (B) and mitochondrial function parameters (C) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild-type or Zeb1ΔM/ΔM mice after sequential treatment of compounds modulating mitochondrial function (n = 4).

D Ultrastructural appearance of mitochondria in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice. Scale bar, 100 nm.

E ATP levels of BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).

F RhoA activity of wild-type and Zeb1ΔM/ΔM osteoclasts cultured on plastic (n = 6).

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using unpaired Student's t test (A, C, F) or two-way ANOVA with Bonferroni correction (E). ns, not significant; **P < 0.01.
A Zeb1/MtCK1 Metabolic Axis Controls Osteoclast Activation and Skeletal Remodeling (2023)
Figure 6

Figure 6. Mitochondrial creatine kinase activity drives mitochondrial bioenergetics.

A A schematic diagram of the mock vector, wild-type human MtCK1, and the metabolic inactive MtCK1C316G mutant expression vector.

B Wild-type BMDMs were transduced with wild-type human MtCK1, differentiated into osteoclasts, and stained with HA (green), MtCK1 (red), and Tomm20 (cyan). Scale bar, 20 μm.

C Wild-type BMDMs were transduced with either a mock vector, wild-type human MtCK1, or a catalytically-inactive MtCK1C316G mutant expression vector, differentiated into osteoclasts, and cell lysates collected for MtCK1, Ckb, Tomm20, VDAC, Zeb1, c-Src, and Ctsk immunoblotting (n = 3).

D Mitochondrial creatine kinase activity of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic (n = 3).

E PCr/Cr ratio of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic substrata as determined by LC/MS analysis (n = 3).

F ATP levels of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts (n = 3).

G Wild-type BMDMs were transduced with lentiviral vectors expressing wild-type MtCK1, a catalytically-inactive MtCK1C316G mutant, or a mock control and differentiated into osteoclasts, and RhoA activity assessed (n = 6).

H, I OCR profile plot (H) and mitochondrial function parameters (I) analyzed by XF Cell Mito Stress Assay in mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).

J, K Mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type pre-osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (J). The actin ring area per cell and resorption pit area were quantified (K; n = 6). Scale bar, upper 20 μm, lower 100 μm.

Data information: Bars and error bars represent mean ± SEM. Data are representative of at least 3 independent experiments with biological replicates. Data were analyzed using one-way ANOVA with Bonferroni correction (D-G, I, K). ns, not significant; *P < 0.05, **P < 0.01.

Source data are available online for this figure.