EMBO/soda-nel huggingface dataset:
Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy (2020)
Figure 4

Figure 4: TCHP contributes to autophagosome maturation

A, The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.

B, Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).

C, Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).

Data information: statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD.
Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy (2020)
Figure 5

Figure 5: p62 accumulation in ECs from patients with CAD and Tchp knock-out mice

A, Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject).

B, Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).

C, Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO). D, migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).

E, Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).

Data information: Data are represented as mean ± SD.

Expanded View Figure Legends
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 1

Figure 1. p53EE is deficient for DNA binding and target gene activation.

A p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin-3a (Nutlin) for 16 hours. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53-/- or p53EE/EE MEFs. For p53EE/EE MEFs three independent replicates are shown.

B De novo motif search using MEME-Chip was performed on the 468 p53 binding peaks (as in A). The best hit motif is reported with corresponding E-value and logo (upper part). Graphs depict a CentriMo enrichment analysis for the best MEME motif (middle) and for known transcription-factor binding sites (bottom). The top two hits are shown with corresponding E-values.

C RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM).

D,E RNAseq data were subjected to gene set enrichment analysis (GSEA). Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin-treated MEFs with the indicated p53 genotypes. (E) Summary of GSEA results for p53-related gene sets. NES, normalized enrichment score; nom P, nominal P value.

F Reverse transcription quantitative PCR (RTqPCR) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg/ml doxorubicin (Doxo). Shown are expression values normalized to β-actin as mean ± SD (n=6). P values were calculated by 2way ANOVA with Sidak's multiple comparisons test.

G-H Western blots of protein lysates prepared from MEFs treated for 24 hours with (G) 10 µM Nutlin or (H) 0.4 µg/ml Doxo.
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 2

Figure 2. p53EE fails to induce apoptosis, cell-cycle arrest, and senescence.

A Proliferation of primary MEFs. Cells were treated o/n with 0.2 µg/ml doxorubicin (Doxo) and pulse-labeled with 32 µM 5-Bromo-2-deoxyuridine (BrdU), fixed and processed for flow cytometry analysis. n=4.

B Long-term proliferation assay with primary MEFs of indicated genotypes. +/+ and -/-: n=3; EE/EE: n=6.

C MEFs were immortalized with the adenoviral oncogene E1A.12S (E1A MEF) and treated with 0.4 µg/ml Doxo for 17 hours. Apoptosis (Annexin V) was quantified by flow cytometry. +/+ and -/-: n=3; EE/EE: n=6. Western blots show expression of E1A and β-actin as loading control.

D Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 µM dexamethasone as a control for p53-independent apoptosis. Cell survival relative to untreated samples was analyzed using CellTiter Glow assay (Promega). n=11 for each time point and genotype.

E mRNA expression analysis (RTqPCR) of p53 target genes in thymocytes 6 hours after 6 Gy irradiation. Shown are expression values normalized to β-actin.

F-I Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL) and (G) proliferation (BrdU); scale bars 50 µm. Red arrowheads highlight TUNEL-positive apoptotic cells. (H,I) Quantification for n=3 mice/genotype (150 crypts/mouse).

Data information: All data are shown as mean ± SD. P values were calculated by 2way ANOVA with Sidak's multiple comparisons test.
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 3

Figure 3. Lethality of p53EE embryos in the absence of Mdm2.

A Observed and expected genotype distribution of newborn offspring from mating Mdm2+/-Trp53EE/+ mice (total number of pups n=185; contingency test P=0.0029).

B Observed and expected genotype distribution of newborn offspring from mating Mdm2+/-Trp53+/- mice (total number of pups n=435; contingency test P=0.912).

C Representative picture of an E9.5 Mdm2-/-Trp53EE/EE embryo (right) displays characteristic phenotypic abnormalities compared to an Mdm2+/-;Trp53EE/EE embryo (left).

D Phenotype analysis of Trp53EE/EE embryos with different Mdm2 genotypes reveals developmental defects in Mdm2-/-Trp53EE/EE embryos starting from E9.5.

E IHC for p53 and cleaved caspase-3 shows strong accumulation of p53EE protein and massive apoptosis in Mdm2-/-Trp53EE/EE compared to Mdm2+/-;Trp53EE/EE control embryos.
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 4

Figure 4. Pharmacological inhibition of Mdm2 unleashes cytotoxic activities of p53EE.

A Mdm2-/-Trp53-/- (double knock-out, DKO) MEFs were transduced with pInducer20-p53EE lentivirus to enable tetracyclin (Tet)-inducible expression of mouse p53EE. Induction of p53EE sensitized cells to apoptosis induced by 24 hour treatment with 1 µg/ml doxorubicin (Doxo) as detected by flow cytometry for Annexin V.

B mRNA expression of p53 target genes was measured in cells from (A) relative to β-actin by RTqPCR following treatment with Tet ± 1 µg/ml Doxo. Expression of the pro-apoptotic p53 target genes Bbc3 (Puma) and Bax is not significantly induced. n=6.

C Proliferation of MEFs with indicated genotypes was analyzed by live-cell imaging in the presence of 10 µM nutlin-3a (Nutlin) and/or 0.05 µg/ml doxorubicin (Doxo). Shown is the median confluence ±SEM (n=12).

D-E Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0.4 µg/ml Doxo. n=3.

F Western blot of E1A-MEFs with indicated genotypes treated with 10 µM Nutlin ± 0.4 µg/ml Doxo for 18 hours reveals induction of apoptosis (cCasp-3, cleaved caspase-3; cParp, cleaved Parp) in double-treated p53EE/EE MEFs in the absence of p53 target gene (p21, Mdm2) activation. *, 10 hours 0.4 µg/ml Doxo; **, 10 hours 0.2 µg/ml Doxo.

G mRNA expression of p53 target genes was measured in E1A MEFs of indicated genotype relative to β-actin by RTqPCR following combined treatment with 10 µM Nutlin and 1 µg/ml Doxo. n=12.

H Western blot of p53EE/EE E1A-MEFs with indicated genotypes treated for 18 hours with 10 µM Nutlin, 0.4 µg/ml Doxo and 30-100-500 nM Ganetespib as indicated. Comb, combined treatment with 3 drugs for 18 hours. Pre, pre-treatment with ganetespib for 24 hours followed by combined treatment with Nutlin and Doxorubicin for 18 hours.

I Western blot of cellular fractions from unstressed and Doxo-treated MEFs of indicated genotype identifies p53EE protein in the mitochondrial fraction. PCNA, Tom20/Bak are shown as nuclear and mitochondrial marker proteins.

Data information: All data are shown as mean ± SD unless indicated otherwise. Significance was tested between the two cell lines or treatments. P values were calculated by 2way ANOVA with Sidak's multiple comparisons test.
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 5

Figure 5. Residual Apoptotic Activities of Mutant p53 in Human Cancer Cells.

A-B Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo. This effect is enhanced by addition of Nutlin and blocked by the pan-caspase inhibitor QVDOph, but not the ferroptosis inhibitor Ferrostatin-1 (Ferr) as control. (A) n=3; (B) n=2.

C Apoptosis analysis using Annexin V staining in cells from (A). n=3.

D mRNA expression analysis (RTqPCR) of p53 target genes following treatment with 0.5 µg/ml Doxo ± 10 µM Nutlin. KO, tet-induced H1299-pInd cells; EE, tet-induced H1299-pInd-p53EE cells; WT, H460 cells. mRNA expression was normalized to β-actin; n=3.

E Proximity ligation assay for p53 with Tom20 in indicated untreated (Mock) or Doxo-treated (0.1 µg/ml, 24 hours) H1299 cells following tet-induction. Shown are representative cells with PLA signals (red) counterstained with DAPI (blue).

F Quantification of proximity ligation assays for p53EE with Tom20, Bcl-2, Bcl-xL and Bak in H1299 cells. Doxo treatment (0.1 µg/ml, 24 hours) as indicated. Plotted is the average number of PLA signals per cell for 10 fields of view.

G Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide. Treatment with the mitochondrial depolarizer CCCP is shown as a positive control; n=3.

H Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet-inducible expression of the indicated human p53 mutants; n=3.

I mRNA expression analysis (RTqPCR) of p53 target genes normalized to β-actin; n=3.

Data information: All data are shown as mean ± SD. Significance was tested by 2way ANOVA with Sidak's multiple comparisons test.
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 6

Figure 6. p53EE fails to suppress tumor development.

A Kaplan-Meier survival plots for mice with indicated genotypes, number of mice, and median survival. Right panel, analysis of survival differences using the Log-rank (Mantel-Cox) test.

B Spectra of malignant tumors found in mice with indicated genotypes (p53-/- n=16; p53EE/- n=24; p53EE/EE n=37; p53+/- n=28; p53EE/+ n=17).

C p53 immunohistochemistry (IHC) in representative spontaneous tumors; scale bars 25 µm.

D Kaplan-Meier survival plots for Eµ-Myc transgenic mice with different p53 genotype.

E Kaplan-Meier survival plots for wild-type recipient mice transplanted with AML1/ETO9a + NrasG12D-transduced fetal liver cells (5 donors/genotype).

F-G p53 IHC for representative samples from (D) and (E) illustrates constitutive stabilization of p53EE in cancer cells; scale bars 25 µm.

Data information: Survival differences were analyzed using the Log-rank (Mantel-Cox) test. Multiple survival curves were compared by ordinary ANOVA with Tukey's multiple comparisons test.
Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses (2019)
Figure 7

Figure 7. p53EE confers survival benefit under chemotherapy.

A Apoptosis in Eµ-Myc lymphoma cells of indicated genotype was measured by flow cytometry (FITC-VAD-FMK staining) at indicated time points after treatment with 3 µg/ml MAF. Significance was calculated versus untreated. n≥5.

B Enrichment of p53 shRNA-expressing Eµ-Myc;p53EE lymphoma cells under MAF treatment. Non-silencing control shRNA served as control. n=3.

C mRNA expression of p53 target genes was measured relative to β-actin by RTqPCR following treatment of Eµ-Myc lymphoma cells of indicated genotype with 3 µg/ml MAF. n≥3.

D-E Kaplan-Meier survival plots for cyclophosphamide-treated versus untreated control mice transplanted with Eµ-Myc lymphoma cells of indicated genotype at day 0. Treatment started ~10 days later when peripheral lymph nodes became palpable. The time course of the experiment is illustrated in a scheme. (E) Summary of data from (D). Survival differences were analyzed using the Log-rank (Mantel-Cox) test.

F Residual disease in the spleen of mice from (D) was quantified 24 hours and 7 days after therapy using a quantitative PCR assays with primers specific to the Eµ-Myc transgene (present only in lymphoma cells). Shown is the copy number of the Eµ-Myc transgene relative to a control locus present in all cells. Untreated Eµ-Myc mice (Myc tg) were used as positive control. Non-transgenic mice (no Myc tg) served as negative controls to define the detection limit. Significance was tested by t-test (two-sided, unpaired).

G Representative bioluminescence imaging (BLI) pictures of mice transplanted with AE9+Nras AML cells. Day 0 indicates the start of combination therapy with cytarabine (AraC) + doxorubicin (Doxo).

H Kaplan-Meier survival plots for animals from (G). Statistical significance of differences calculated by Log-rank (Mantel-Cox) test between treated Eµ-Myc;p53EE/EE AML and all other groups is indicated in the table. C, untreated control; T, therapy (AraC+Doxo).

Data information: All data are shown as mean ± SD. Significance was tested by 2way ANOVA with Sidak's multiple comparisons test unless indicated otherwise.

Expanded View Figure Legends
Inositol-Requiring Enzyme-1 Regulates Phosphoinositide Signaling Lipids and Macrophage Growth (2020)
Figure 1

Figure 1. Inhibition of IRE1 RNase activity leads to significant reduction in PI(3,4,5)P3 and downstream growth signaling.

(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).

(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).

(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.

(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).

Data information: All data are mean ± SEM; (n=3). Unpaired t-test with Welch's correction. *P≤ 0.05, **P≤ 0.01.
Inositol-Requiring Enzyme-1 Regulates Phosphoinositide Signaling Lipids and Macrophage Growth (2020)
Figure 2

Figure 2. IRE1-dependent changes in miRNA expression in ER-stressed macrophages

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform.

(A) Table showing fold change (log2) of statisticaly significant (p-value <0.050) top PA-upregulated miRNAs in IRE1+/+ and IRE1-/- cells. NS indicates micro RNAs that didn't show statistically significant difference. Fold change was calculated by comparing mean intensity values of micro array signal for vehicle treatment with PA treatment (n=3 biological replicates).

(B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.

(C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).

(D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).

(E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).

Data information: All data are mean ± SEM (n=3); unpaired t-test with Welch's correction; *P≤ 0.05).
Inositol-Requiring Enzyme-1 Regulates Phosphoinositide Signaling Lipids and Macrophage Growth (2020)
Figure 3

Figure 3. miR-2137 is regulated by IRE1 RNase activity in vivo

(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).

(C, D) (C) C57BL/6 (WT) and (D) Apoe-/- mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (WT; n=4-4 mice per group, Apoe-/-; n=5-5 mice per group).

(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).

Data information: Data are mean ± SEM; unpaired t-test with Welch's correction. *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001.
Inositol-Requiring Enzyme-1 Regulates Phosphoinositide Signaling Lipids and Macrophage Growth (2020)
Figure 4

Figure 4. miR-2137 is regulated by IRE1 RNase activity

(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).

(B) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).

(C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).

(D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).

(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).

(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).

(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.

(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).

Data information: All data are mean ± SEM (n=3); unpaired t-test with Welch's correction. *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001.
Inositol-Requiring Enzyme-1 Regulates Phosphoinositide Signaling Lipids and Macrophage Growth (2020)
Figure 5

Figure 5. INPPL1 is a target for miR-2137

(A) Putative miR-2137 binding site on 3` UTR of human (Gene ID 3636), chimpanzee (Gene ID 451403), rat (Gene ID 65038) and mouse (Gene ID 16332) INPPL1 mRNA.

(B) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates).

(C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates).

(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).

Data information: All data are mean ± SEM (n=3); unpaired t-test with Welch's correction. *P≤ 0.05, **P≤ 0.01.