Journal: eLife

Article Title: NMDA receptors in visual cortex are necessary for normal visuomotor integration and skill learning

doi: 10.7554/eLife.71476

Figure Lengend Snippet: ( A ) We injected an adeno-associated viral vector (AAV) to express Cre recombinase unilaterally and another to express a calcium indicator bilaterally (GCaMP6f) in V1 of ΔGrin1 mice. ( B ) Experimental timeline: a first group of Grin1 mice (ΔGrin1 juv ) was dark-reared from birth. We injected an AAV to express Cre at postnatal day (P)21 unilaterally in V1, injected a second AAV bilaterally to express GCaMP6f, and implanted imaging windows bilaterally at P30. A second group of Grin1 mice (ΔGrin1 adult ) was reared normally and received the same injections at p>100. All mice then had six sessions of visuomotor exposure in a closed-loop (CL) virtual environment before imaging experiments. ( C ) Example two-photon images showing co-expression of GCaMP6f and Cre-mCherry constructs. ( D ) In situ hybridization against Grin1 mRNA (see Materials and methods) confirming the local knockout of Grin1 in V1. Blue: hematoxylin stain for cell nuclei; brown: Grin1 hybridization signal. Brain regions were identified using a mouse brain atlas . ( E ) Injection sites of Cre were readily visible in Grin1 in situ hybridization images. Outside of the injection site (i), labeling was dense in most cells with multiple puncta per cell. In injection sites (ii), labeling was almost completely absent. Inset shows a cell with one punctum (arrow); if a cell had more than two of these puncta, it was counted positive in the analysis shown in ( F ). ( F ) The fraction of cells positive (more than two puncta per cell) for Grin1 mRNA in 0.5 mm × 0.5 mm regions in injection sites (Δ) was strongly reduced compared to regions outside of injection sites (C). ( G ) Grin1 knockout reduced expression of Grin1 in all major cortical neuron types. Left: violin plots of the number of single-cell sequencing mRNA reads corresponding to the portion of the Grin1 gene knocked out in the ΔGrin1 mice and control mice. Layer 2/3 (L2/3), layer 4 (L4), layer 5 (L5), and layer 6 (L6) excitatory neurons, and parvalbumin (PV)-positive, somatostatin (SST)-positive, and vasoactive peptide (VIP)-positive interneurons. Expression levels are normalized to the total number of reads per nuclei. Right: the same data for ΔGrin1 mice. Expression levels were significantly downregulated in all neuron types, with the exception of VIP neurons (see for statistics).

Article Snippet: For mapping viral expression, an mCherry sequence (Addgene 237633) was amended as a separate chromosome.

Techniques: Injection, Plasmid Preparation, Imaging, Expressing, Construct, In Situ Hybridization, Knock-Out, Staining, Hybridization, Labeling, Sequencing, Control

Journal: PLOS Pathogens

Article Title: Bacterial infection promotes tumorigenesis of colorectal cancer via regulating CDC42 acetylation

doi: 10.1371/journal.ppat.1011189

Figure Lengend Snippet: (A) CDC42 K153 acetylation is regulated by SIRT family deacetylases. HEK293T cells transfected with Flag-CDC42 were treated with the deacetylase inhibitors TSA (2 μM) and NAM (10 mM) for 16 h before harvesting. K153 acetylation was determined by IP/WB. (B) SIRT2 decreases K153 acetylation in vivo . Overexpression of SIRT2 significantly decreases K153 acetylation. Flag-CDC42 was co-transfected with or without HA-SIRT2 into HEK293T cells, and K153 acetylation was analyzed by IP with anti-Flag antibody and WB with anti-K153Ac antibody. (C) SIRT2 decreases K153 acetylation in vitro . Purified GST-CDC42 protein was incubated with the cell lysate of HEK293T cells expressing HA-SIRT2 or the control vector. K153 acetylation was determined by the GST pull-down assay, followed by WB with anti-K153Ac antibody. (D) A reduced acetylation of K153 was shown in AK7-treated cells. Flag-CDC42 was co-transfected with or without HA-SIRT2, followed by treatment with the SIRT2 specific inhibitor AK7 (10 μM) or dimethyl sulfoxide (DMSO) (as a negative control) for 16 h. K153 acetylation was then determined by IP/WB. SIRT2 knockdown increases K153 acetylation. Flag-CDC42 was transfected with or without HA-PAK4 into HEK293T cells with SIRT2 knocked down, and the cells were infected with S . Typhimurium. Salmonella infection failed to reduce K153 acetylation and binding between CDC42 with PAK4 in the SIRT2 knockdown cells. Cell lysates were used for IP with anti-Flag antibody. K153 acetylation was then analyzed by WB with anti-K153Ac antibody (E), and the association between CDC42 and PAK4 was determined by WB (F).

Article Snippet: Anti-CDC42 (#10155-1-AP, 1:1000 for WB), anti-PAK4 (#14685-1-AP, 1:1000 for WB), anti-PAK1(#21401-1-AP, 1:1000 for WB), monoclonal anti-GST(#66001-2-Ig, 1:10000 for WB), polyclonal anti-HA (#51064-2-AP, 1:5000 for WB), monoclonal anti-SIRT2 (#66410-1-IG, 1:10000 for WB), anti-beta tubulin (#10094-1-AP, 1:2000 for WB), MMP-2 (#10373-2-AP, 1:1000 for WB), MMP-9 (#10375-2-AP, 1:1000 for WB) were purchased from Proteintech; Monoclonal anti-HA (#AB0025, 1:5000 for WB); E-cadherin (#BS1098, 1:1000 for WB) were from Bioworld; Monoclonal anti-CDC42 (#sc-8401, 2 μg per 500 μg of total protein for Immunoprecipitation), anti-p-PAK4 (sc-135775, 1:200 for WB) were from Santa Cruz; Anti-p-PAK1 (#2601, 1:1000 for WB), anti-p-AKT (#4060, 1:1000 for WB), anti-AKT (#4691, 1:1000 for WB), anti-p-ERK (#9101, 1:1000 for WB), anti-ERK (#4370, 1:1000 for WB), anti-p-p38 (#9211, 1:1000 for WB), anti-p38 (#9212, 1:1000 for WB), anti-p-JNK (#9251, 1:1000 for WB), anti-ERK (#9252, 1:1000 for WB), anti-caspase 3 (#14220, 1:1000 for WB) and anti-PARP (#9542, 1:1000 for WB) were from Cell Signaling Technology; Monoclonal anti-HA (#M180, 1:500 for Immunoprecipitation), monoclonal anti-Flag (#M185, 1:500 for Immunoprecipitation), polyclonal anti-Flag (#PM020, 1:1000 for WB) and deacetylase inhibitors TSA (#9950) were from MBL; Deacetylase inhibitor NAM (#N1651) was from APExBIO; CBP/p300 inhibitor A-485 (#N1651) was from MCE; SIRT2 inhibitor AK7 (#4754–10) was from Tocris Bioscience; Staurosporine (#abs810006) was from Absin; Polybrene (#H9268) and puromycin (#P8833) were from Sigma-Aldrich.

Techniques: Transfection, Histone Deacetylase Assay, In Vivo, Over Expression, In Vitro, Purification, Incubation, Expressing, Control, Plasmid Preparation, Pull Down Assay, Negative Control, Knockdown, Infection, Binding Assay

Journal: PLOS Pathogens

Article Title: Bacterial infection promotes tumorigenesis of colorectal cancer via regulating CDC42 acetylation

doi: 10.1371/journal.ppat.1011189

Figure Lengend Snippet: (A) Human CRC specimens were confirmed by HE staining, and the expression levels of CDC42 and CDC42 K153 acetylation and the localization of bacteria in tissues of 17 human CRC specimens were detected by IHC. CDC42 K153 acetylation level was significantly lower in the colorectal adenocarcinoma tissues than in the adjacent normal colorectal tissues as determined by IHC. The average of IHC intensity ± SD were quantitated by modified H-score from two groups of 17 patient samples (B) and 69 patients with different CRC stages (C) is shown. (D) Kaplan–Meier analysis of overall survival of 69 patients with CRC according to CDC42 K153 acetylation level. (E) Model of CDC42 K153 acetylation effect on colorectal tumorigenesis. Under physiological conditions, acetylated CDC42 K153 can maintain the normal physiological function of cells through MAPK phosphorylation (including ERK, JNK and p38) mediated by the CDC42-PAK4 signaling pathway. When Salmonella infects a host cell, SIRT2 is activated to deacetylate CDC42 K153, which causes an impaired binding of its downstream effector PAK4 and an attenuated phosphorylation of p38 and JNK, consequently reduces cell apoptosis. Moreover, low acetylation level of CDC42 K153 may contribute to the migration and invasion abilities of CRC cells, and promoting tumorigenesis, which may activate tumors mainly through the CDC42-PAK signaling axis.

Article Snippet: Anti-CDC42 (#10155-1-AP, 1:1000 for WB), anti-PAK4 (#14685-1-AP, 1:1000 for WB), anti-PAK1(#21401-1-AP, 1:1000 for WB), monoclonal anti-GST(#66001-2-Ig, 1:10000 for WB), polyclonal anti-HA (#51064-2-AP, 1:5000 for WB), monoclonal anti-SIRT2 (#66410-1-IG, 1:10000 for WB), anti-beta tubulin (#10094-1-AP, 1:2000 for WB), MMP-2 (#10373-2-AP, 1:1000 for WB), MMP-9 (#10375-2-AP, 1:1000 for WB) were purchased from Proteintech; Monoclonal anti-HA (#AB0025, 1:5000 for WB); E-cadherin (#BS1098, 1:1000 for WB) were from Bioworld; Monoclonal anti-CDC42 (#sc-8401, 2 μg per 500 μg of total protein for Immunoprecipitation), anti-p-PAK4 (sc-135775, 1:200 for WB) were from Santa Cruz; Anti-p-PAK1 (#2601, 1:1000 for WB), anti-p-AKT (#4060, 1:1000 for WB), anti-AKT (#4691, 1:1000 for WB), anti-p-ERK (#9101, 1:1000 for WB), anti-ERK (#4370, 1:1000 for WB), anti-p-p38 (#9211, 1:1000 for WB), anti-p38 (#9212, 1:1000 for WB), anti-p-JNK (#9251, 1:1000 for WB), anti-ERK (#9252, 1:1000 for WB), anti-caspase 3 (#14220, 1:1000 for WB) and anti-PARP (#9542, 1:1000 for WB) were from Cell Signaling Technology; Monoclonal anti-HA (#M180, 1:500 for Immunoprecipitation), monoclonal anti-Flag (#M185, 1:500 for Immunoprecipitation), polyclonal anti-Flag (#PM020, 1:1000 for WB) and deacetylase inhibitors TSA (#9950) were from MBL; Deacetylase inhibitor NAM (#N1651) was from APExBIO; CBP/p300 inhibitor A-485 (#N1651) was from MCE; SIRT2 inhibitor AK7 (#4754–10) was from Tocris Bioscience; Staurosporine (#abs810006) was from Absin; Polybrene (#H9268) and puromycin (#P8833) were from Sigma-Aldrich.

Techniques: Staining, Expressing, Bacteria, Modification, Binding Assay, Migration

Journal: bioRxiv

Article Title: A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

doi: 10.1101/2021.08.17.456253

Figure Lengend Snippet: A , Overall proportion of different alternative splicing events in control and Ago2 cKO meiotic and post-meiotic germ cells. B , Change in exon percent spliced in (PSI) for individual transcripts with a significant difference in skipped exons in Ago2 cKO compared to control germ cells. C , Nuclear:cytoplasmic ratios in post-meiotic cells for two transcripts bound ( Ak7 , Hmgb2 ) and two transcripts not bound ( Ift27 , Ppib ) by nuclear AGO2. Localization was assessed by single-molecule RNA in situ hybridization (RNAscope) in Ago2 cKO compared to control germ cells. Data points show ratios for individual cells from at least three tubules. Bars show mean and 95% confidence interval. **p<0.01, Welch’s t-test. Sample images are shown in Figure S4C .

Article Snippet: Immunoblotting was performed using the following primary antibodies and dilutions: Histone H3 (Abcam, ab1791, 1:40000 or Abcam, ab18521, 1:1000), AGO2 (Abcam, ab186733, 1:2000 or Abcam, ab32381, 1:1000), H3K9me3 (Abcam, ab8898, 1:4000), HMGB2 (Abcam, ab124670, 1:3000), DAZL (Abcam, ab34139, 1: 2000), YBX2 (Abcam, ab154829, 1:4000), AK7 (Novus Biologicals, NBP2-92176, 1: 3000), GTSF1 (NOVUS Biologicals, NBP1-83934,1:1000), PTBP2 (Proteintech, 55186-1-AP, 1:4000), α-TUBULIN (Santa Cruz, sc-8035, 1:1000), SYCP3 (Abcam, ab97672, 1:1000), ACRV1 (guinea pig, gift of Dr. Prabhakara Reddi, University of Illinois , 1:5000).

Techniques: Alternative Splicing, Control, RNA In Situ Hybridization, RNAscope

Journal: Aging (Albany NY)

Article Title: AK7-deficiency reversal inhibits ccRCC progression and boosts anti-PD1 immunotherapy sensitivity

doi: 10.18632/aging.206006

Figure Lengend Snippet: Expression profile of AK7 in tumor. ( A ) Expression of AK7 mRNA in different cancers and corresponding normal tissues. ( B ) The expression of AK7 mRNA in ccRCC was significantly higher than that in normal kidney tissue. ( C – G ) Differences in SPC25 mRNA expression depending on stage, grade, nodal metastasis status, subtype and race. ( H ) Expression of AK7 in normal renal tissues and ccRCC tissues. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were cultured in a 5% CO 2 chamber at 37°C with RFMI-1640 (Gibco, USA) containing 10% FBS (NEWZERUM, Newzerum, Christchurch, New Zealand) and 1% P-S. si-AK7 and si-NC were produced by KeyGEN BioTECH (Jiangsu, China).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: AK7-deficiency reversal inhibits ccRCC progression and boosts anti-PD1 immunotherapy sensitivity

doi: 10.18632/aging.206006

Figure Lengend Snippet: AK7 knockdown promoted the proliferation, invasion and migration ability of human ccRCC cell lines. ( A , B ) Three siRNAs (si1, si2, and si3) were designed to silence AK7 in ccRCC cells (786-O and Caki-1), and validated by qRT-PCR. ( C , D ) The growth curves of 786-O ( C ) and Caki-1 ( D ) cells were plotted after transfection with si1-AK7/si-NC based on CCK-8 assay. ( E , F ) Colony formation assays demonstrated that knockdown of AK7 promoted the proliferation of 786-O and Caki-1 cells. ( G – I ) Transwells experiment demonstrated that knockdown of AK7 expression could effectively promote the migration and invasion ability of ccRCC cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were cultured in a 5% CO 2 chamber at 37°C with RFMI-1640 (Gibco, USA) containing 10% FBS (NEWZERUM, Newzerum, Christchurch, New Zealand) and 1% P-S. si-AK7 and si-NC were produced by KeyGEN BioTECH (Jiangsu, China).

Techniques: Knockdown, Migration, Quantitative RT-PCR, Transfection, CCK-8 Assay, Expressing

Journal: Aging (Albany NY)

Article Title: AK7-deficiency reversal inhibits ccRCC progression and boosts anti-PD1 immunotherapy sensitivity

doi: 10.18632/aging.206006

Figure Lengend Snippet: Overexpression of AK7 inhibited proliferation, invasion and migration of human ccRCC cell lines. ( A , B ) qRT-PCR verified the efficiency of overexpression of AK7 in 786-O and AKI-1 cell lines. ( C , D ) The growth curves of 786-O ( C ) and Caki-1 ( D ) cells were plotted after overexpression of AK7 based on CCK-8 assay. ( E , F ) Colony formation assays demonstrated that overexpression of AK7 inhibited the proliferation of 786-O and Caki-1 cells. ( G – I ) Transwells experiment demonstrated that overexpression of AK7 could effectively inhibit the migration and invasion ability of ccRCC cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were cultured in a 5% CO 2 chamber at 37°C with RFMI-1640 (Gibco, USA) containing 10% FBS (NEWZERUM, Newzerum, Christchurch, New Zealand) and 1% P-S. si-AK7 and si-NC were produced by KeyGEN BioTECH (Jiangsu, China).

Techniques: Over Expression, Migration, Quantitative RT-PCR, CCK-8 Assay

Journal: Aging (Albany NY)

Article Title: AK7-deficiency reversal inhibits ccRCC progression and boosts anti-PD1 immunotherapy sensitivity

doi: 10.18632/aging.206006

Figure Lengend Snippet: AK7 can be used as a prognostic indicator and a predictor of immunotherapy effect in ccRCC patients. ( A ) In pancarcinoma, patients with high expression of AK7 have a better prognosis than those with low expression. ( B ) In ccRCC, patients with high expression of AK7 had longer OS than those with low expression. ( C ) In ccRCC at stage 4, patients with high expression of AK7 had longer OS than those with low expression. ( D – F ) In patients treated with anti-PD1 ( D ), anti-PD-L1 ( E ), and anti-CTLA-4 ( F ), high expression of AK7 has a better prognosis. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were cultured in a 5% CO 2 chamber at 37°C with RFMI-1640 (Gibco, USA) containing 10% FBS (NEWZERUM, Newzerum, Christchurch, New Zealand) and 1% P-S. si-AK7 and si-NC were produced by KeyGEN BioTECH (Jiangsu, China).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: AK7-deficiency reversal inhibits ccRCC progression and boosts anti-PD1 immunotherapy sensitivity

doi: 10.18632/aging.206006

Figure Lengend Snippet: The expression of AK7 was correlated with immunosuppressive factors. ( A ) Correlation between AK7 and immunoinhibitors in different cancers. ( B ) The expression of AK7 was negatively correlated with the expression of CTLA4, TIGIT, IL10, PD1, IL0RB, LAG3 and other immunosuppressive factors. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were cultured in a 5% CO 2 chamber at 37°C with RFMI-1640 (Gibco, USA) containing 10% FBS (NEWZERUM, Newzerum, Christchurch, New Zealand) and 1% P-S. si-AK7 and si-NC were produced by KeyGEN BioTECH (Jiangsu, China).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: AK7-deficiency reversal inhibits ccRCC progression and boosts anti-PD1 immunotherapy sensitivity

doi: 10.18632/aging.206006

Figure Lengend Snippet: Overexpression of AK7 inhibits RCC growth and enhances anti-PD1 efficacy. ( A ) Schematic diagram of establishment of mouse subcutaneous tumor model in each group. ( B ) Three lentiviruses were designed to overexpress AK7 expression in RENCA cell lines, and their overexpression efficiency was verified by qRT-PCR. ( C ) Images of subcutaneous tumors in each group. ( D , E ) Analysis of subcutaneous tumors in the respective groups. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were cultured in a 5% CO 2 chamber at 37°C with RFMI-1640 (Gibco, USA) containing 10% FBS (NEWZERUM, Newzerum, Christchurch, New Zealand) and 1% P-S. si-AK7 and si-NC were produced by KeyGEN BioTECH (Jiangsu, China).

Techniques: Over Expression, Expressing, Quantitative RT-PCR