Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RT-PCR analysis of the mutually exclusive α-exons in Mef2d transcript using total RNA from the indicated fetal hind limb muscle and tissues from adult wild-type mice. ( B ) RT-PCR analysis of Mef2d α exons using soleus RNA from line 2. The numbers indicate PSI; data are mean ± SD; n = 3. The numbers indicate the percent spliced in (PSI). Bolded numbers are significant by Student T test. ( C ) RT-PCR analysis of the alternative exons in Mef2c and Mef2a transcripts using total RNA from gastrocnemius muscle from line 1. Data are mean ± SD; n = 3. PSI for exon is indicated and was not significantly different between the genotypes by Student’s t test. ( D ) Western blot showing MEF2D protein levels in TA (upper left) and Soleus muscles (upper right) from line 2. The bottom panels show Coomassie stained blots, from the upper panels showing total protein loaded. The numbers are mean ± SD; n = 3. Student’s t test found no differences in genotypes. ( E ) RT-qPCR showing relative mRNA levels Mef2d , Mef2a , and Mef2c relative to Rpl30 using total RNA in the indicated muscle groups from line 2 mice. Data are mean ± SEM; n = 4. ** P = 0.0088 (multiple Student’s t test, unpaired). .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Staining, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RT-PCR analysis of the mutually exclusive α-exons and alternative β exon in Mef2d transcript using total RNA from the indicated muscle groups from Wt and Mef2dα2 Eko mice from line 1. The numbers indicate the percent spliced in (PSI) for indicated exon. Data are mean ± SD; n = 3. Bold numbers indicate significance by Student t test. ( B ) Western blot showing MEF2D protein levels in TA (upper left) and Soleus muscles (upper right) from line 1. Bottom panels show Coomassie stained blots from the upper panels showing total protein loaded. The numbers indicate the relative protein level normalized to total protein by Coomassie stained blots. Data are mean ± SD; n = 3. ( C ) RT-qPCR showing mRNA levels Mef2d , Mef2a , and Mef2c relative to Rpl30 using total RNA in the indicated muscle groups from line 1 mice. Data are mean ± SEM; n = 3. No significant changes were found between the genotypes by multiple t test in ( B , C ). .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Muscles, Staining, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RNA-sequencing data using poly-A selected RNA from WT and MEF2Dα2 Eko quadriceps muscles were aligned to mm10 UCSC browser showing Mef2d α exons region. ( B ) Graph showing number of genes that were down or upregulated in MEF2Dα2 Eko muscles in comparison to age and sex-matched WT muscles (FDR < 0.05). ( C ) RT-qPCR showing relative expression of Bdh1, Oxct1, and Acat1 transcripts normalized to Hprt transcript levels in WT and MEF2Dα2 Eko soleus muscles from line 1. Data are mean ± SEM; n ≥ 6. ** P < 0.01, *** P < 0.001 (Student’s t test). The exact P value is indicated above the bar graphs. ( D ) Western blot showing BDH1 level in soleus muscles of indicated mice from line 1. The panel on the right show BDH1, OXCT1, and ACAT1 level when normalized to total protein loaded as estimated by Coomassie staining of the same blot. Data are mean ± SEM, n = 3, * P < 0.016, ** P < 0.0042, **** P = 0. 000082 (Student’s t test). .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: RNA Sequencing, Muscles, Comparison, Quantitative RT-PCR, Expressing, Western Blot, Staining

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) β-hydroxybutyrate (BHB) tolerance test in untrained 28-week-old male sedentary mice. Graphs displays mean ± SEM, with n ≥ 9. * P < 0.05 ( P = 0.02525 at 45 min, P = 0.021921 at 60 min), ** P < 0.01 ( P = 0.002777 at 90 min) by multiple t test between genotypes at different time-points. ( B ) The indicated age-matched male mice were run for 55 min at 18 m/min, and BHB was measured immediately after running (0 h), and 1–3 h post-run. The pre-run BHB values are from the same mice a day before the experiment. Data are mean ± SD, n = 10, n.s. not significant; P > 0.05, ** P < 0.01 ( P = 0.00121 at 0 h), * P < 0.05 ( P = 0.0485 at 2-h post-exercise), *** P < 0.001 (0.000233088 at 3-h post-exercise) by multi p le t test between genotypes at different time-points. ( C ) The indicated age-matched male mice were fed control or ketogenic diet for 2-weeks and BHB was measured without fasting. Data are mean ± SD; n ≥ 8. The exact P value as calculated by one-way ANOVA is indicated above the bar graphs. ( D ) Comparison between state 3 JO 2 (ADP 200 µM) for isolated mitochondria from WT and MEF2Dα2 Eko skeletal muscles in presence of alpha-ketoglutarate (AKG) and AKG+Acetoacetate (AcAc). The representative time courses for these experiments are shown in Fig. . Data are mean ± SD; n = 5. The exact P value as calculated by one-way ANOVA is indicated above the bar graphs. ( E ) ChIP quantitative PCR for indicated genes using MEF2D and Serotype control (IgG) are shown (left), representative DNA product visualized on a 5% acrylamide gel (right). Data are mean ± SD; n = 4. For Bdh1, actual P value is indicated; for others, * P < 0.05, ** P < 0.01 (paired Student’s t test). The exact P value is indicated above the bar graphs. .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Control, Comparison, Isolation, Muscles, Real-time Polymerase Chain Reaction, Acrylamide Gel Assay

Journal: EMBO Reports

Article Title: The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice

doi: 10.1038/s44319-025-00578-3

Figure Lengend Snippet: ( A ) RT-qPCR showing relative expression of indicated transcripts normalized to Hprt transcript levels using total RNA from WT and MEF2Dα2 Eko mice livers. Data are mean ± SEM, n = 5, Student’s t test found no differences in genotypes. ( B ) Representative time-courses of isolated mitochondrial respiration for WT and MEF2Dα2 Eko mice transitioning from state 1 to state 4 respiration under AKG± AcAc. The respiratory rates are expressed as nmol/min/mg mitochondrial protein. The transitions from state 1 to state 4 respiration were monitored by first adding isolated mitochondria (0.05 mg/mL) to the respiration buffer at t = 0 min leading to state 1. At t = 2 min, substrates were added to energize the mitochondria, which led to state 2 respiration. This was followed by sequential additions of incremental ADP concentrations (100 and 200 µM). AKG: Alpha-ketoglutarate and AcAc: acetoacetate. ( C ) MEF2D ChIP data from Gönczi et al viewed on IGV viewer (version 2.19.4) for Igfn1, Bdh1, Oxct1, and Acat1 gene locus. The bi-directional arrowed line shows the region where we designed our primers for our analyses. .

Article Snippet: MEF2D (ChIP) , Proteintech , 14353-1-AP.

Techniques: Quantitative RT-PCR, Expressing, Isolation

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: RUNX1 Interacts with the AF4-MLL Complex and Activates Gene Targets (A) RUNX1 ChIP-seq in SEM cells compared with MLL-C:H3K4Me3 and MLL-N:AF4-C:H3K79Me2 ChIP-seq. (B–D) Sample ChIP-seq tracks from SEM cells across MEF2C (B), ADAM10 (C), and SPI1/PU.1 (D). (E) Gene expression analysis by real-time PCR in SEM cells treated with two different RUNX1 siRNAs (gray bars, siRNA#1; white bars, siRNA#2). For each experiment, the PCR signal was quantified relative to control-treated cells. Results represent the mean ± SD of three independent knockdown experiments. (F) Western blots as indicated in SEM cells treated with a nontargeting control, RUNX1 siRNA#1, or a wild-type MLL siRNA. (G) RUNX1 protein complex interactions. RUNX1 can interact with a wild-type AF4 complex (interaction 1), a wild-type MLL complex (interaction 2), and potentially with an AF4-MLL complex (interaction 3). (H and I) Immunoprecipitation (IP) experiments using RS4;11 (H) and SEM (I) nuclear extracts. Extracts were IP’d with αIgG (lane 2), αAF4-N (lane 3), αRUNX1 (lane 4) or αMLL-C (lane 5), blotted and probed with the antibodies indicated. Input lanes represent 1% of the amount of extract used for the IPs. (J) A schematic of the MEF2D , JUNB , JUND, and SPI-1 (aka PU.1 ) loci showing the approximate location of PCR primer sets (open arrow heads) used for ChIP analysis. Black box indicates consensus RUNX1 binding motifs in the upstream regulatory region (URE) of SPI-1 ( Huang et al., 2008 ; Huang et al., 2011 ) and the first intron of MEF2D ( Pencovich et al., 2011 ). Gray box indicates exon 1 of MEF2D , JUND , JUNB, and SPI-1 . (K–M) ChIP analysis in SEM cells treated with a nontargeting control or RUNX1 siRNA#1 at the targets as indicated using antibodies to AF4-N (K), MLL-C (L), and RBBP5 (M). Error bars represent the ±SD of three separate PCR reactions. See also and Figure S5 .

Article Snippet: Complexes were eluted by boiling in NuPAGE gel loading buffer (Life Technologies) for 5 min. Antibodies The following antibodies were used with the indicated techniques: H3K4Me3 (Active Motif, 39159, ChIP and ChIPseq); H3K79Me2 (Millipore, 04-835, ChIP and ChIPseq); MLL-N (Bethyl, A300-086A, ChIP and ChIPseq); MLL-C (Bethyl, A300-374A, Western blot); MLL-C (Active Motif, 61295, IPs and ChIP), AF4-C (Abcam, ab31812, ChIP, ChIPseq and Western blot); AF4-N (Bethyl, A302-344A, ChIP, IP and Western blots); ENL (Bethyl, A302-268A, ChIP and Western blots); AF9 (Bethyl, A300-595A, ChIP and Western blots); CyclinT1 (Bethyl, A303-496A, ChIP and Western blots); aff4 (Bethyl, A302-538A, ChIP); Menin (Bethyl, A300-105A, Western blots); RUNX1 (Cell Signaling, 4334 for IPs and Western blots, Abcam, ab23980 antibodies for IPs and ChIPseq); HOXA9 (Millipore, 07-178, Western blots); GAPDH (Bethyl, A300-641A); RbBP5 (Bethyl, A300-109A, Western blots); WDR5 (Bethyl, A302-430A, Western blots); MEF2C (Cell Signaling, 5030, Western blots); LEF1 (Bethyl, A303-486, Western blots); ADAM10 (Abcam, ab1997, Western blots); ZEB2 (Bethyl, A302-474A, Western blots); SPI-1/PU.1 (Cell Signaling, 2258S, Western blots); ELL2 (Bethyl, A302-505A, Western blots); MEF2D (Bethyl, A303-521A, Western blots); JUNB (Bethyl, A302-704A, Western blots); JUND (Cell Signaling, 5000, Western blots); SPI-B (Abcam, ab42436 Western blots), βActin (Sigma, A4700).

Techniques: ChIP-sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Control, Knockdown, Western Blot, Immunoprecipitation, Binding Assay

Journal: Cell Reports

Article Title: RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

doi: 10.1016/j.celrep.2012.12.016

Figure Lengend Snippet: RUNX1 ChIPseq and AF4-MLL Complex Data, Related to Figure 6 (A) Sample ChIP-seq tracks from SEM cells across RUNX1 , HOXA9 , MEF2D and JUND . (B) ChIP-seq overlap between the RUNX1 SEM cell target gene set versus the set of RUNX1 target genes from ( Pencovich et al., 2011 ). (C) Western blots for the proteins indicated in SEM cells treated with a scrambled control or an MLL-AF4 siRNA. Proteins were detected using the antibodies indicated except MLL-AF4, which was detected with an AF4-C antibody. (D) Gene expression analysis of selected genes in THP-1 cells treated with RUNX1 siRNA (siRNA#1). For each experiment, the PCR signal was quantified relative to the appropriate control treated cells. Results represent the average of three independent knockdown experiments, and error bars represent the standard deviation between experiments. (E) A schematic of protein complex interactions centering on the RUNX1 protein. RUNX1 can interact with a wild-type AF4 complex (interaction 1) through CyclinT1 ( Elagib et al., 2008 ; Jiang et al., 2005 ), a wild-type MLL-C complex (interaction 2) and potentially with an AF4-MLL complex (interaction 3). (F) Immunoprecipitation (IP) experiments using RS4;11 (t(4;11)), SEM (t(4;11)) and CCRF-CEM (wild-type MLL1) nuclear extracts. Extracts were IP’d with either αAF4-N (lane 3, 6 and 9) or a control αIgG (lane 2, 5 and 8) antibody, blotted and probed with the antibodies indicated. Lane 1,4 and 7 (Inputs) represents 1% of the amount of extract used for the IPs. AF4-MLL is indicated by a white arrowhead (AF4-MLL is 328 KDa, the white arrowhead represents the ∼194KDa Taspase 1 cleaved product) while wild-type AF4 is indicated by a black arrowhead (wild-type AF4 has a predicted size of 131 KDa but an apparent MW of 175 KDa). MLL-C is the Taspase 1 cleaved product of both AF4-MLL and wild-type MLL which is 134KDa but runs with an apparent MW of 180KDa. (G) Real Time PCR of MEF2D , JUNB and SPI-1 expression in SEM, RS4;11 and CCRF-CEM cells. (H) A schematic of the MEF2D , JUNB and SPI-1 (aka PU.1 ) loci showing the approximate location of PCR primer sets (open arrow heads) used for ChIP analysis. Black box = consensus RUNX1 binding motifs in the upstream regulatory region (URE) of SPI-1 ( Huang et al., 2008 ; Huang et al., 2011 ). and the first intron of MEF2D ( Pencovich et al., 2011 ). Grey box = exon1 of MEF2D , JUNB and SPI-1 . (I and J) ChIP analysis in SEM (black bars), RS4;11 (gray bars), CCRF-CEM (white bars) at the targets as indicated using antibodies to AF4-N (I) or RUNX1 (J). (K) Real Time PCR expression of AF4-MLLder4a and der4b ( Kumar et al., 2011 ) in SEM cells treated with a scrambled control (purple bars), an AF4-MLL siRNA (siRNA#10, blue bars) or an AF4-MLL siRNA (siRNA-K, orange bars) from ( Kumar et al., 2011 ). Error bars represent the ± SD of three separate PCR reactions. In each individual experiment, control values were arbitrarily set to 100. AF4-MLL siRNA sequences are in supplemental methods. (L) Western blots of the AF4-MLL knockdowns in (K) using the antibodies indicated. The apparent MW of AF4-MLL and MLL-C is explained in (F) above. (M) Western blots at day 4 and day 8 of SEM cells treated with both AF4-MLL siRNA-K and siRNA#10 at day 0, day 2, day 4 and day 7. Antibodies are as indicated. AF4-MLL is indicated by a white arrowhead while wild-type AF4 is indicated by a black arrowhead as explained in (F) above.

Article Snippet: Complexes were eluted by boiling in NuPAGE gel loading buffer (Life Technologies) for 5 min. Antibodies The following antibodies were used with the indicated techniques: H3K4Me3 (Active Motif, 39159, ChIP and ChIPseq); H3K79Me2 (Millipore, 04-835, ChIP and ChIPseq); MLL-N (Bethyl, A300-086A, ChIP and ChIPseq); MLL-C (Bethyl, A300-374A, Western blot); MLL-C (Active Motif, 61295, IPs and ChIP), AF4-C (Abcam, ab31812, ChIP, ChIPseq and Western blot); AF4-N (Bethyl, A302-344A, ChIP, IP and Western blots); ENL (Bethyl, A302-268A, ChIP and Western blots); AF9 (Bethyl, A300-595A, ChIP and Western blots); CyclinT1 (Bethyl, A303-496A, ChIP and Western blots); aff4 (Bethyl, A302-538A, ChIP); Menin (Bethyl, A300-105A, Western blots); RUNX1 (Cell Signaling, 4334 for IPs and Western blots, Abcam, ab23980 antibodies for IPs and ChIPseq); HOXA9 (Millipore, 07-178, Western blots); GAPDH (Bethyl, A300-641A); RbBP5 (Bethyl, A300-109A, Western blots); WDR5 (Bethyl, A302-430A, Western blots); MEF2C (Cell Signaling, 5030, Western blots); LEF1 (Bethyl, A303-486, Western blots); ADAM10 (Abcam, ab1997, Western blots); ZEB2 (Bethyl, A302-474A, Western blots); SPI-1/PU.1 (Cell Signaling, 2258S, Western blots); ELL2 (Bethyl, A302-505A, Western blots); MEF2D (Bethyl, A303-521A, Western blots); JUNB (Bethyl, A302-704A, Western blots); JUND (Cell Signaling, 5000, Western blots); SPI-B (Abcam, ab42436 Western blots), βActin (Sigma, A4700).

Techniques: ChIP-sequencing, Western Blot, Control, Gene Expression, Knockdown, Standard Deviation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Binding Assay

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Transcription factors MEF2A and MEF2D regulate MTORC1 activity upon amino acid stimulation. (A) Gene set enrichment analysis of the MTOR pathway gene list in HeLa cells with overexpression of MEF2-VP16 versus controls by using RNA-seq data. (B) HeLa cells transfected with either dn MEF2 or vector as control were treated with amino acid (aa) starvation and amino acid restimulation with or without troin1 (250 nM). Western blot analysis of phosphorylation of RPS6KB1, EIF4EBP1 and AKT is shown. Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (middle) and p-AKT:AKT (bottom) ratios. (C) HeLa cells transfected with MEF2-VP16 or vector were treated and then analyzed by immunoblotting similar to (B). (D) HeLa cells transfected for 48 h with indicated siRNA were starved of amino acids and restimulated before immunoblotting analysis. Right plots show the p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. (E) HeLa cells that stably knocked-down both MEF2A and MEF2D (sh MEF2A/D ) or control (NC) were starved for amino acids and restimulated with increasing levels (expressed as % of the concentration in DMEM medium) of leucine. Lysates were analyzed for MTORC1 activity as in (D). Bottom plots show the p-RPS6KB1:RPS6KB1 (right), p-EIF4EBP1:ACTB (left) ratios. (F) MEF2A and MEF2D double-knockdown HeLa cells transfected with MEF2A or/and MEF2D and then subjected to amino acid administration. MTORC1 activity was confirmed by immunoblotting. Plots on the right show p-RPS6KB1:RPS6KB1 (top) and p-EIF4EBP1:ACTB (bottom) ratios. Data are presented as the mean ± S.E.M. ( n = 3 independent experiments, two-sided Student’s t-test for B, C and E, one-way analysis of variance [ANOVA] for D, two-way ANOVA post hoc test for F, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant compared with indicated group).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Activity Assay, Over Expression, RNA Sequencing, Transfection, Plasmid Preparation, Control, Western Blot, Phospho-proteomics, Stable Transfection, Concentration Assay, Knockdown

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: MEF2A and MEF2D are required for MTORC1 mediated protein, lipid synthesis and inhibit autophagy. (A) HeLa cells depletion of both MEF2A and MEF2D by indicated siRNA were treated with amino acid administration in the presence or absence of 10 μg/ml puromycin (PURO) and cycloheximide (CHX). Cell lysates were analyzed by western blot with an antibody to puromycin (17H1). Right plots show the ratio of PURO:ACTB. (B) MEF2A and MEF2D double-knockdown HeLa cells with or without transiently re-expressing MEF2A and MEF2D after utilized TSC2 -targeting (si TSC2 ) or control (si NC ) siRNA for 48 h under normal culture condition. Oil red O staining was performed to quantify intracellular lipid levels. Scale bar: 20 μm. (C) control or shRNA-mediated depletion of MEF2A and MEF2D HeLa cells were treated with the starvation of amino acids for 1 h and then restimulated with amino acids for indicated time. Phospho-ULK1 (Ser757), ULK1, LC3B-II levels were analyzed by western blotting. Right plot shows the quantification of LC3B-II:GAPDH. Short exposure (SE), long exposure (LE). (D) MEF2A and MEF2D knockdown and control HeLa cells that stably express GFP-LC3 were starved of amino acids for 1 h and then restimulated with amino acids for indicate duration and stained with LysoTracker. Time-lapse images were taken. Scale bar: 5 μm. (E) MEF2A and MEF2D double-knockdown and control HeLa cells were transfected for 24 h with GFP-RFP-LC3 adenovirus. After that, cells were subjected to amino acid starvation for 4 h and restimulation for 30 min. Autophagy flux was tested by confocal. Plot on bottom shows the ratios of colocalization of GFP and RFP. Scale bar: 10 μm. (F) statistical analysis of the cell diameters of control HeLa cells and the cells that depletion of MEF2A and MEF2D by using a cell counter. Data are presented as the mean ± S.E.M. ( n = 3 independent experiments for A, B and C, n = 3 independent fields per condition for E, n = 10 per group for F. two-sided Student’s t-test for A, C, D and E, one-way ANOVA for B and E, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant compared with indicated group).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Western Blot, Knockdown, Expressing, Control, Staining, shRNA, Stable Transfection, Transfection

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: MEF2A and MEF2D positive regulate MTORC1 translocation to lysosomes. (A) MEF2A and MEF2D double-knockdown and control HeLa cells transfected with Flag-tagged TMEM192 for 24 h were subjected to amino acid starvation and restimulation. Purified lysosomes via lyso-IP assay (methods). Protein levels of MTOR and RPTOR (MTORC1 components), LAMP1 and LAMP2 (lysosome), CALR (endoplasmic reticulum), VDAC (mitochondria), GOLGA2 (Golgi) and RPS6KB1 (cytosol) were confirmed by immunoblotting. Right graph shows the quantification result of MTOR after normalization for LAMP1. (B) cells as in (A) were starved of and restimulated with amino acids for the indicated times before being analyzed by immunofluorescence and quantified to calculate the percentage of colocalization coefficient of MTOR (green) and lysosomal protein LAMP2 (red). Scale bar: 10 μm. (C) MEF2A and MEF2D double-knockdown HeLa cells were subjected to amino acid starvation and restimulation paradigm. Cell lysates were immunoprecipitated with anti-RRAGC antibody. Immunoprecipitates or total cell lysate (TCL) samples were probed for MTOR, RPTOR and RRAGC. Right graph shows the quantification result of MTOR after normalization for RRAGC. (D) MEF2A and MEF2D double-knockdown and control HeLa cells that either transfected with constitutively active RRAG GTPases (Flag-tagged RRAGA Q66L and RRAGC S75N ) or vector coimmunostained for lysosomal marker LAMP2 (green), MTOR (red) and Flag-tag (violet). Cells were starved of amino acid and restimulated with amino acids before processing and imaging. The graph on the bottom shows the colocalization coefficient. Scale bar: 10 μm. (E) control (lanes 1–2) and knockdown of both MEF2A and MEF2D (lanes 3–6) HeLa cells that transfected with indicated RRAG GTPases were then treated with amino acids administration. Phosphorylation and protein levels of RPS6KB1 and EIF4EBP1 were determined by immunoblotting. Right graph shows the RPS6KB1:RPS6KB1 ratios. Data are presented as the mean ± S.E.M. ( n = 3 independent experiments for A, C and E, n = 4 independent fields per condition for B, D. two-sided Student’s t-test for A, B, C and E, one-way ANOVA for D, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Translocation Assay, Knockdown, Control, Transfection, Purification, Western Blot, Immunofluorescence, Immunoprecipitation, Plasmid Preparation, Marker, FLAG-tag, Imaging, Phospho-proteomics

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: MEF2A and MEF2D control MTORC1 lysosome localization and activation via transcriptional regulation of FNIP1 and FNIP2. (A) heatmap of the binding sites of MEF2s at the positions − 3.0 kb upstream to + 3.0 kb downstream relative to the transcription start site (TSS). (B) volcano plot showing DEGs in control and MEF2-VP16 overexpression HeLa cells. The significant changed upregulated genes were labeled with gene names, NR4A1 and ARC are well-characterized substrates of MEF2s. (C) venn diagrams displaying the overlap of downregulated and upregulated genes from the transcriptome with MEF2 bound genes. Bottom table shows FNIP2 and other genes which are well known as MEF2s substrates including Fos/c-fos , NR4A1 , ZNF122 and ARC in the subset of overlap of upregulated genes with MEF2 bound genes. (D) left-schematic shows the predicted MEF2 binding sites by conserved MEF2 consensus at the positions upstream to FNIP1 and FNIP2 genes transcription start site (TSS). Right plots show the PCR results revealed by chromatin immunoprecipitation assay. MEF2D_1,2 indicate the ChIP-PCR results by different antibodies (1-BD biosciences, 610774; 2-CST, 77986). (E) control (lanes 1–2) and MEF2A and MEF2D double-knockdown (lanes 3–8) HeLa cells that either transfected with indicated plasmids were starved of and restimulated with amino acids. Phosphorylation and protein levels of RPS6KB1 and EIF4EBP1 were determined by immunoblotting, as indicated. Plots on the right show p-RPS6KB1:RPS6KB1 (top) and p-EIF4EBP1:ATCB (bottom) ratios. (F) MEF2A and MEF2D double-knockdown HeLa cells stably overexpressing FNIP1 or FNIP2 by pLVX-IRES-ZsGreen constructs were starved of and restimulated with amino acids before coimmunostained of LAMP2 and MTOR and analyzed by confocal. MTOR (green), LAMP2 (red), ZsGreen-FNIP1 (violet) and ZsGreen-FNIP2 (violet). The plot on the bottom shows the colocalization coefficient. Scale bar: 10 μm. Data are presented as the mean ± S.E.M. (n = 3 independent experiments for E. n = 4 independent fields per condition for F. one-way ANOVA for F, two-way ANOVA for E **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Control, Activation Assay, Binding Assay, Over Expression, Labeling, Chromatin Immunoprecipitation, Knockdown, Transfection, Phospho-proteomics, Western Blot, Stable Transfection, Construct

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: SRC directly interacts with and phosphorylates MEF2D upon mitogenic stimulation. (A) lysate derived from human embryonic kidney 293T (HEK293T) cells transfected as indicated were immunoprecipitated with IgG or anti-hemagglutinin (HA) antibody. Immunoprecipitation (IP) and TCL were probed for indicated antibodies. (B) GST affinity isolation assay was performed using HEK293T cells purified Flag-tagged MEF2D protein and bacterially purified GST or GST-tagged SRC (method), followed by immunoblotting with indicated antibodies. (C) HeLa cells were cultured in a serum free medium for 4 h, then treated with or without EGF for 30 min. Cell lysates were immunoprecipitated with anti-MEF2D antibody and blotted with anti-SRC antibody (top panel). TCL was probed for indicated antibodies. (D) HEK293T purified Flag-tagged MEF2D protein was pre-treated with lambda PP and then incubated with commercial active GST-tagged SRC kinase in a kinase assay buffer, followed by immunoblotting with indicated antibodies. (E) HEK293T cells transfected as indicated were immunoprecipitated with anti-HA antibody and blotted with pan p -Tyr antibody. TCL was analyzed by immunoblotting for indicated antibodies. (F) HEK293T cells transfected HA-tagged MEF2D with either Flag-tagged SRC -WT (wild-type) or Flag-tagged SRC -KD (SRC K298M , a kinase-dead form of SRC). Cell lysates were immunoprecipitated with anti-HA antibody. IP and TCL samples were probed for indicated antibodies. (G) HeLa cells were maintained in a serum free medium for 4 h, and then treated with or without serum, EGF or serum plus EGF for 30 min. Total cell lysates and endogenous MEF2D immunoprecipitated were analyzed by immunoblotting with indicated antibodies. Actin as a loading control. (H) HeLa cells transfected with indicated siRnas against SRC (in two RNAi sequences) or control ( siNC ) for 48 h were subjected to serum free culture for 4 h, followed by stimulation with or without EGF for 30 min. Cells were lysed and subjected to immunoprecipitation with anti-MEF2D antibody, followed by immunoblotting with indicated antibodies. (I) HeLa cells were subjected to serum free medium for 4 h, and then either untreated or restimulation with EGF in the presence or absence of SRC kinase inhibitor Dasatinib or SU6656. Endogenous MEF2D was immunoprecipitated, followed by immunoblotting with indicated antibodies.

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Derivative Assay, Transfection, Immunoprecipitation, Isolation, Purification, Western Blot, Cell Culture, Incubation, Kinase Assay, Control

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: SRC-mediated tyrosine phosphorylation of MEF2D is required for transcriptional activity of MEF2D and in regulation of MTORC1 activity. (A) His-tagged MEF2D- WT or MEF2D mutant constructs (mutation of Tyr33, 57, 69, 72, 117, 131, 225, 333, 337 and 478 residues) were co-transfected with or without Flag-tagged SRC in HEK293T cells for 24 h. Cells were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with p -tyr antibody. (B) HEK293T cells transfected His-tagged MEF2D- WT or MEF2D- 3YF mutants with or without Flag-tagged SRC were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with indicated antibodies. (C) Flag-tagged MEF2D-WT or its 3YF mutant protein purified from HEK293T cells was incubated with commercial active GST-tagged SRC kinase in a kinase assay buffer, followed by immunoblotting with p -tyr antibody. (D) sequence alignment of the residues flanking across different species. Black arrowheads point to the tyrosine residues corresponding to the Tyr333 and tyr 337 residues in human MEF2D. (E) HeLa cells that transfected with Flag-tagged MEF2D- WT or MEF2D- 3YF were maintained in a serum free medium for 4 h, followed with or without EGF treatment. Cell lysates were prepared and immunoprecipitation were analyzed by immunoblotting. (F) luciferase assay was performed in depletion of both MEF2A and MEF2D HeLa cells after co-transfection of indicated expression plasmids and wild-type ( MEF2 reporter-WT) or mutated ( MEF2 reporter-mt) luciferase reporter plasmids for 24 h. (G) qRT-PCR analysis was performed in MEF2A and MEF2D double-knockdown HeLa cells that reconstructed with MEF2D- WT or MEF2D- 3YF. The mRNA levels of FNIP1 , FNIP2 , FLCN , NR4A1 , ZMAT4 and DAAM1 were shown. (H) MEF2A and MEF2D double-knockdown HeLa cells were starved with serum for 4 h and then treated with EGF for 3 h before immunoblotting analysis of the activation of MTORC1 with indicated antibodies. Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. (I) MEF2A and MEF2D double-knockdown HeLa cells that transfected with indicated plasmids were subjected to serum starvation for 4 h and restimulated with EGF for 3 h. MTORC1 activity was analyzed similarly to (H). Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. Data are presented as the mean ± S.E.M. (n = 3 independent experiments. two-sided Student’s t-test for H, one-way ANOVA for I, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Phospho-proteomics, Activity Assay, Mutagenesis, Construct, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, Kinase Assay, Sequencing, Luciferase, Cotransfection, Expressing, Quantitative RT-PCR, Knockdown, Activation Assay

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: MEF2D promotes pancreatic cancer cell proliferation and correlates with clinical and pathological parameters. (A) pancreatic cancer cell line AsPC-1 (top) and PANC-1 (bottom) that depletion of MEF2A and MEF2D , transfected with either Flag-tagged MEF2D- WT or MEF2D- 3YF, respectively and subjected to the cell proliferation assay (CCK8) to determine viable cell number. NC and knockdown of both MEF2A and MEF2D group co-transfected with empty vector. (B) the colony formation assay was performed with MEF2A and MEF2D double-knockdown AsPC-1 cells (left) or PANC-1 cells (right). The cells were maintained in complete medium containing 10% FBS for two weeks before staining and imaging. Scale bars: 5 mm. (C) Representative images (left) and weights (right) of xenograft tumors from nude mice implanted with MEF2A and MEF2D double-knockdown AsPC-1 cells versus control cells. n = 10 mice per group. (D) volumes of tumor burden in nude mice implanted with MEF2A and MEF2D double-knockdown or control AsPC-1 cells were measured after implantation. n = 10 mice per group. (E) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of MEF2D mRNA expression. Overall survival was compared between these two groups, as shown in Kaplan-Meier curves. Log-rank P values are indicated. (F) immunoblot analysis of MEF2D, FNIP1, FNIP2, EIF4EBP1 and p-EIF4EBP1 were performed in pancreatic tumors and paired adjacent normal tissues. ACTB was probed for as a loading control. Tumor-adjacent normal tissue (N), tumor (T). (G) randomly selected pancreatic cancer samples and their paired adjacent normal tissues were lysed and subjected to IP against MEF2D, followed by immunoblotting with the pan p -Tyr antibody. (H) Representative immunohistochemistry of pancreatic cancer and paired adjacent normal tissue sections were stained for MEF2D, FNIP1, FNIP2 and p-EIF4EBP1 respectively. Scale bars: 200 μm. Data are presented as the mean ± S.E.M. (n = 3 independent experiments for a and B, n = 8 biologically independent samples for F, n = 4 biologically independent samples per group for G and H. two-sided Student’s t-test for a and D, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phospho-EIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine/p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phospho-SRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Transfection, Proliferation Assay, Knockdown, Plasmid Preparation, Colony Assay, Staining, Imaging, Control, Expressing, Western Blot, Immunohistochemistry