Journal: Journal of Cellular and Molecular Medicine

Article Title: The roles of α V integrins in lens EMT and posterior capsular opacification

doi: 10.1111/jcmm.12213

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: cMaf sc7866 , 1% BSA in PBS , 1 hr at RT 1:100 dilution , Santa Cruz.

Techniques: Blocking Assay, Incubation

Journal: Journal of molecular endocrinology

Article Title: Cap-Independent mRNA Translation Is Upregulated in Long-Lived Endocrine Mutant Mice

doi: 10.1530/JME-19-0021

Figure Lengend Snippet: A) Representative western blot of target protein levels in HEK cells transfected with control vector, or with YTHDF1 or YTHDF2 vectors. B) Bars represent the mean ± S.E.M.SEM from 8 independent transfections. All values were normalized to 1 for the respective controls. C) CT values of qRT-PCR performed using mRNA purified from at least 8 independent transfections per group. The (*) indicates statistical significance (p<0.05) relative to controls. D) The bar represents the mean ± S.E.M. from at least 8 independent transfections, showing treatment with or without rapamycin (Rapa+ or Rapa-) in enhancing the effects of YTHDF on protein expression. (*) indicates statistical significance (p<0.05) relative to rapamycin untreated cells (Rapa -) from same group.

Article Snippet: Clones corresponding to human YTHDF1-Flag and YTHDF2-Flag were purchased from Origene (Rockville.

Techniques: Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR, Purification, Expressing

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: Enhanced learning and memory in DF2‐CKO mice. A) Representative images of co‐immunostaining for YTHDF2 with NeuN (upper panel) or GFAP (down panel) in the mouse hippocampus. YTHDF2 predominantly colocalized with the neuronal marker NeuN, and sparsely with the astrocytic marker GFAP. B) Representative immunostaining images of YTHDF2 in control and DF2‐CKO mice, showing efficient knockout of YTHDF2 expression in both the hippocampus and cortex. C) Representative images of adult (10 weeks old) control and DF2‐CKO mice. D) Body weight curves of control and DF2‐CKO mice from born to 8 weeks old ( n = 7 mice per group, two‐way ANOVA, F (1, 96) = 0.363, p = 0.548), indicating no significant difference in body weight between groups. E) Schematic representation of the protocol used to test fear memory. F) The freezing level curves of control and DF2‐CKO mice during fear conditioning ( n = 18 mice per group, two‐way ANOVA, Control vs DF2‐CKO: F (1, 34) = 1.006, p = 0.323). G,H) Contextual (G) and auditory (H) fear memory assessed at 1 h after fear training (G: n = 18 mice per group, unpaired two‐tailed t ‐test, t 34 = 2.422, p = 0.021; H: n = 9 mice per group, unpaired two‐tailed t ‐test, t 16 = 0.677, p = 0.508). I,J) Contextual (I) and auditory (J) fear memory assessed 24 h after fear training (I: n = 18 mice per group, unpaired two‐tailed t ‐test, t 34 = 3.611, p = 0.001; J: n = 9 mice per group, unpaired two‐tailed t ‐test, t 16 = 0.018, p = 0.986). K) The Barnes maze test was used to assess spatial memory. The upper panel shows the training protocol, and the lower panel shows heat maps of movement trajectories for control and DF2‐CKO mice on the test day (day 8). L) Performance of control and DF2‐CKO mice in training sessions of Barnes maze test ( n = 15, 18 mice per group, two‐way ANOVA, group: F (1, 186) = 2.026, p = 0.156). M) Latencies (time to locate the escape box) in the probe trial of the Barnes Maze test ( n = 15, 18 mice per group, unpaired t ‐test, t 31 = 3.101, p = 0.004). N) The time spent in target quadrant and other quadrants during the probe trial of the Barnes Maze test ( n = 15, 18 mice per group, two‐way ANOVA, F (3, 124) = 11.1, p < 0.001, post hoc: Bonferroni's test, target: p < 0.001, left: p = 0.040, opposite: p = 0.053, right: p > 0.999).

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Immunostaining, Marker, Control, Knock-Out, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: Reexpression of YTHDF2 in the adult hippocampus reverses the memory performance. A) Schematics of AAV constructs overexpressing YTHDF2 (AAV– YTHDF2 ) or GFP (AAV–GFP) (up panel) and Illustration of bilateral viral injections into the mouse hippocampus (down panel). ITR, inverted terminal repeats; CMV, cytomegalovirus promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. B) Representative fluorescence images of the mouse hippocampus after AAV infection. C) Representative western blot (up panel) and quantification data (down panel) of YTHDF2 expression in mice hippocampus injected with AAV– YTHDF2 ( n = 4 mice per group, one‐way ANOVA, F (3, 12) = 77.56, p < 0.001, post hoc: Bonferroni's test, Control + AAV–GFP vs Control + AAV– YTHDF2 , p = 0.001, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– YTHDF2 , p < 0.001, Control + AAV–GFP vs DF2‐CKO + AAV–GFP, p < 0.001). D) The freezing curves of control and DF2‐CKO mice injected with AAV during fear conditioning ( n = 14, 13, 11, 12 mice, two‐way ANOVA, F (3, 184) = 0.642, p = 0.589). E,F) Contextual (E) and auditory (F) fear memory assessed 1 h after fear training ( n = 14, 13, 11, 12 mice, E: one‐way ANOVA, F (3, 46) = 5.245, p = 0.003, post hoc: Bonferroni's test, Control + AAV–GFP vs Control + AAV– YTHDF2 , p = 0.027, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– YTHDF2 , p = 0.306, Control + AAV–GFP vs DF2‐CKO + AAV–GFP: p > 0.999, Control + AAV–GFP vs DF2‐CKO + AAV– YTHDF2 : p > 0.999; F: one‐way ANOVA, F (3, 46) = 2.377, p = 0.082). G,H) Contextual (G) and auditory (H) fear memory assessed 24 h after fear training ( n = 14, 13, 11, 12 mice, G: one‐way ANOVA, F (3, 46) = 10.49, p <0.0001, post hoc: Bonferroni's test, Control + AAV–GFP vs Control + AAV– YTHDF2 , p = 0.043, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– YTHDF2 , p = 0.017, Control + AAV–GFP vs DF2‐CKO + AAV–GFP, p = 0.026, Control + AAV–GFP vs DF2‐CKO + AAV– YTHDF2 : p > 0.999; H: one‐way ANOVA, F (3, 46) = 1.956, p = 0.134). I) Schematic diagram illustrating the experimental timeline of AAV– YTHDF2 or AAV–GFP injection followed by fear conditioning and subsequent patch‐clamp recording. J) Representative traces of sEPSCs. K,L) Cumulative probability plots and bar graph (inside) indicated both decreased sEPSCs frequency (K) and amplitude (L) in hippocampal CA1 pyramidal neurons from DF2‐CKO mice hippocampus injected with AAV– YTHDF2 after fear conditioning ( n DF2‐CKO+AAV–GFP = 21 cells, 4 mice, n DF2‐CKO+AAV– YTHDF2 = 23 cells, 4 mice; unpaired t ‐test, t 42 = 4.253, p < 0.001, KS test, p < 0.0001 for frequency; unpaired t ‐test, t 42 = 2.337, p = 0.024, KS test, p < 0.0001 for amplitude). M) Representative traces (left) and I–O curves (right) showed the amplitudes of evoked EPSC at CA3–CA1 pathway from DF2‐CKO mice hippocampus injected with AAV–GFP or AAV– YTHDF2 after fear conditioning ( n DF2‐CKO+AAV–GFP = 15 cells, 2 mice, n DF2‐CKO+AAV– YTHDF2 = 13 cells, 2 mice; unpaired t ‐test, t 8 = 3.776, p = 0.005). N) Representative traces (left) and histogram (right) of paired‐pulse ratio (PPR) from DF2‐CKO mice hippocampus injected with AAV–GFP or AAV– YTHDF2 after fear conditioning ( n DF2‐CKO+AAV–GFP = 15 cells, 2 mice, n DF2‐CKO+AAV– YTHDF2 = 14 cells, 2 mice; unpaired t ‐test, t 27 = 3.665, p = 0.001). O) Representative traces (left) and summary plots (right) showed LTP induced by HFS (1 train) at CA3–CA1 pathway in DF2‐CKO mice injected with AAV–GFP or AAV– YTHDF2 after fear conditioning ( n Control = 10 cells from 4 mice, n DF2‐CKO = 10 cells from 4 mice; unpaired two‐tailed t ‐test, t 18 = 18.91, p < 0.0001). Data are presented as mean ± standard error. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Construct, Virus, Fluorescence, Infection, Western Blot, Expressing, Injection, Control, Patch Clamp, Two Tailed Test

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: Loss of YTHDF2 enhances the stability of synaptic function related target mRNAs. A) Venn plot showing the overlap of high confidence YTHDF2 ‐binding target mRNAs between replicate 1 and replicate 2 in YTHDF2 RIP‐seq. Significant targets were defined as foldchange (IP/input) >1.8 and FDR < 0.05. B) Venn plot showing the overlap of total mRNAs from RNA seq after fear conditioning and YTHDF2 RIP‐seq. mRNAs with FPKM ≥ 0.5 in RNA‐seq were included. C) GO enrichment analysis of overlapped genes identified from RNA seq and YTHDF2 RIP‐seq in (B). Top ten enriched BP and CC terms were showed. D) Cumulative distribution and violin plot (inside) showing the distribution of log 2 ‐fold change in nontarget mRNAs and DF2 RIP target mRNAs upon YTHDF2 depletion (Mann–Whitney test, U = 1.034E7, p = 1.56E−14, KS test, Z = 4.767, p < 0.0001). E) Cumulative distribution and violin plot (inside) showing the distribution of log2‐fold change in nontarget mRNAs and DF2 RIP target mRNAs upon YTHDF2 depletion after fear conditioning. (Mann–Whitney test, U = 9986652, p = 6.35E−22, KS test, Z = 4.808, p < 0.0001). F) Cumulative distribution and violin plot (inside) of mRNA half‐life of YTHDF2 targets (1757) in hippocampal primary cultured neurons from either control or DF2‐CKO mice (Mann–Whitney test, U = 1418545, p = 3.24E−5, KS test, Z = 2.176, p = 1.54E−4). G) Cumulative distribution and violin plot (inside) showing the distribution of log 2 ‐fold change of ΔmRNA half‐life between DF2‐CKO and control neurons for nontargets, and YTHDF2 targets (Mann–Whitney test, U = 6084342, p = 5.58E−4, KS test, Z = 3.410, p = 1.59E−10). H) Cumulative distribution plot and violin plot (inside) of the enrichment in YTHDF2 RIP over input for transcripts with 0 (gray), 1–4 (yellow), or 4+ (red) m 6 A‐CLIP‐seq peaks (Kruskal–Wallis test, K = 78.97, p = 7.11E−18, Dunn's test multiple comparison of two or samples, non vs low m 6 A: p = 5.62E−8, non vs high m 6 A: p = 4.36E−13). Data in violin plots are presented as the median and quartiles. *** p < 0.001; **** p < 0.0001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Binding Assay, RNA Sequencing, MANN-WHITNEY, Cell Culture, Control, Comparison

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: The activity‐dependent protein synthesis is enhanced in DF2‐CKO mice hippocampus. A) Cumulative distribution and violin plot (inside) showing the log 2 ‐fold change in protein levels between DF2‐CKO and control mice in hippocampal tissue under basal conditions. Data are stratified by nontargets versus YTHDF2 RIP targets (Mann–Whitney test, U = 840670, p = 0.770, KS test, Z = 0.741, p = 0.642). B) Cumulative distribution and violin plot (inside) showing the log 2 ‐fold change in protein levels between DF2‐CKO and control mice in hippocampal tissue after fear conditioning. Data are stratified by nontargets versus YTHDF2 RIP targets (Mann–Whitney test, U = 728491, p = 6.78E−6, KS test, Z = 2.495, **** p = 7.87E−6). C) Representative blot (left) and quantification data (right) of puromycin signal in KCl‐treated primary cultured hippocampus neurons from either control or DF2‐CKO mice ( n = 7, 5, 7, 5 wells, one‐way ANOVA, F (3, 20) = 12.64, p <0.0001, post hoc: Turkey's test, Control vs Control + KCl, p = 0.047, Control + KCl vs DF2‐CKO + KCl, p = 0.010). D) Cumulative distribution and violin plot (inside) showing the log 2 ‐translational efficiency (TE) of genes in control and DF2‐CKO mice hippocampus at 1 h after fear conditioning (Mann–Whitney test, U = 2.37E7, p = 2.92E−21, KS test, Z = 6.232, p <0.0001). E) Volcano plot showing the significantly changed TE in DF2‐CKO mice compared to control. Significantly changed TE was defined as log 2 TE > 1 and p < 0.05. F) KEGG and GO enrichment analysis of upregulated proteins identified from hippocampus proteomics at 4 h after fear conditioning. Top ten enriched BP and CC terms were showed. Upregulated proteins were defined as fold change (DF‐CKO/Control) > 1.2 and p <0.05. G,H) Representative western blot (left) and quantification data (right) of GluN2B (G) and SEMA4B (H) expression in PSD fraction and total hippocampal lysates from control and DF2‐CKO mice at 4 h after fear conditioning (G: PSD fraction: n = 4 mice per group, unpaired two‐tailed t ‐test, t 6 = 3.036, p = 0.023; total protein: n = 6 mice per group, unpaired two‐tailed t ‐test, t 10 = 0.329, p = 0.749; H: PSD fraction: n = 4 mice per group, unpaired two‐tailed t ‐test, t 6 = 3.600, p = 0.011; total protein: n = 6 mice per group, unpaired two‐tailed t ‐test, t 10 = 2.492, p = 0.032). Data in violin plots are presented as the median and quartiles. Data in bar plots are presented as mean ± standard error. **** p < 0.0001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Activity Assay, Control, MANN-WHITNEY, Cell Culture, Western Blot, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: Knockdown of SEMA4B reverses the phenotype of DF2‐CKO mice. A) IGV visualization of YTHDF2 binding peaks on SEMA4B mRNA in YTHDF2 RIP‐seq. The binding peaks were enriched in IP samples compared to input. RIP1 and RIP2 represented two biological replicates of RIP‐seq. B) RIP‐qPCR analysis shows significant enrichment of SEMA4B , Tanc2 , and Grin2b in YTHDF2 ‐immunoprecipitated samples compared to the negative control RPS29 (not identified as a YTHDF2 target in RIP‐seq), indicating specific binding of YTHDF2 to these transcripts. C) The mRNA level of SEMA4B was analyzed by qPCR in Actinomycin‐D (ActD)‐treated primary neurons from control and DF2‐CKO mice ( n = 3 cell dishes per group, repeated two‐way ANOVA, group factor: F (1, 4) = 11.14, p = 0.029, post hoc: Bonferroni's test, 3 h: p = 0.024). D) MeRIP‐qPCR analysis shows significant enrichment of SEMA4B , TANC2 , and Grin2b in m 6 A‐immunoprecipitated samples compared to the negative control Rps21 (not identified as a m 6 A‐modified target in meRIP‐seq), indicating the presence of m 6 A modification on these transcripts. E) Representative blot (left) and quantification data (right) of SEMA4B expression in hippocampus of mice injected with AAV– SEMA4B shRNA ( n = 6, 3, 5, 4 mice, one‐way ANOVA, F (3, 14) = 8.778, p = 0.002, Bonferroni's test: Control + AAV–GFP vs Control + SEMA4B shRNA, p = 0.013, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– SEMA4B shRNA, p = 0.038). F) The freezing curves of DF2‐CKO mice injected with AAV– SEMA4B shRNA during fear conditioning ( n = 20, 11, 10, 10 mice, repeated two‐way ANOVA, group: F (3, 47) = 2.558, p = 0.066). G,H) Contextual (G) and auditory (H) fear memory assessed 1 h after fear training (G: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 47) = 7.72, p < 0.001, Bonferroni's test: Control + AAV–GFP vs Control + AAV– SEMA4B ShRNA, p = 0.036, Control + AAV–GFP vs DF2‐CKO + AAV–GFP, p = 0.087, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– SEMA4B ShRNA, p = 0.036; H: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 45) = 0.7619, p = 0.521). I,J) Contextual (I) and auditory (J) fear memory assessed 24 h after fear training (I: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 47) = 7.838, p < 0.001, Bonferroni's test: Control + AAV–GFP vs Control + AAV– SEMA4B shRNA, p = 0.442, Control + AAV–GFP vs DF2‐CKO + AAV–GFP, p = 0.005, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– SEMA4B shRNA, p = 0.030; J: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 47) = 0.1705, p = 0.916). K) Representative traces of sEPSCs. L,M) Cumulative probability plots and bar graph (inside) indicated both decreased sEPSCs frequency (L) and amplitude (M) in CA1 pyramidal neurons from DF2‐CKO mice with AAV– SEMA4B expression ( n DF2‐CKO+AAV–GFP = 26 cells, 3 mice, n DF2‐CKO+AAV–shRNA = 21 cells, 3 mice; unpaired t ‐test, t 45 = 2.652, p = 0.011, KS test, p < 0.0001 for frequency; unpaired t ‐test, t 45 = 4.287, p <0.0001, KS test, p < 0.0001 for amplitude). N) Representative traces of sIPSCs. O,P) Cumulative probability plots and bar graph (inside) showing unaffected inhibitory neurotransmission ( n DF2‐CKO+AAV–GFP = 24 cells, 3 mice, n DF2‐CKO+AAV–shRNA = 19 cells, 3 mice; unpaired t ‐test, t 41 = 0.623, p = 0.537 for frequency; unpaired t ‐test, t 41 = 1.505, p = 0.140 for amplitude). Data are presented as mean ± standard error. * p < 0.001; **** p < 0.0001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Knockdown, Binding Assay, Immunoprecipitation, Negative Control, Control, Modification, Expressing, Injection, shRNA