Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet: SKIV2L of the hSKI complex is present at telomeres in G2 (A) Subcellular fractionation assay in asynchronous HeLa1.3 cells. (B) Proteomics of isolated chromatin segments analysis showing the binding of hSKI to telomeres throughout the cell cycle. Tables are listing the number of unique peptide numbers isolated, including the fold change values of unique peptides normalized to the asynchronous values (top panel) and the relative LFQ intensity values identified by PICh. AS: asynchronous. Scr: scramble. (C) WB of hSKI (SKIV2L, TTC37, and WDR61) and SKIV2L2 in shCtr (Control) or shSKIV2L HeLa1.3 cells. (D) Immunofluorescence of pre-extracted cells showing co-localization of SKIV2L and TTC37 with TRF2 in asynchronous (AS) and G2-synchronized cells. % of total number of SKIV2L or TTC37 foci colocalizing with TRF2 are normalized to shCtr AS samples (means ± SEM, n = 2 independent experiments, scale bar 15 μm). t test ∗ p < 0.05, ∗∗∗ p < 0.001. (E) Proximity ligation assay of SKIV2L-TRF2, showing increasing number of foci in G2 synchronized cells (median, Q1 and Q3, 707 (AS) and 638 (G2) cells scored per condition, 3–4 independent experiments, scale bar 10 μm). Mann-Whitney U test ∗∗∗∗ p < 0.0001. (F) ChIP-dot blot of SKIV2L and TRF1 in AS and G2-synchronized shCtr and shSKIV2L cells (means ± SEM, n = 3). t test ∗ p < 0.05, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: The membrane was blocked using non-fat milk 5% (w/v) in PBS-T (PBS 1X; 0.1% (v/v) Tween-20) and subsequently incubated overnight at 4°C with the primary antibody (anti-RNAseH1 (1/500, Abnova, H00246243-M01), anti-GFP (1/5000, Abcam, ab290), anti-β-actin (1/5000, Abcam, ab8226, used as a loading control), anti-H3 (1/1000, Abcam, ab10799), anti-SKIV2L (1/1000, Proteintech group, 11462-1-AP), anti-TTC37 (1/500, Novus Biologicals, NBP1-93640), anti-WDR61 (1/500, Sigma-Aldrich, SAB1401852; 1/1000, Thermofisher PA5-40079), anti-α-Tubulin (1/1000, Sigma-Aldrich, T6199), anti-TRF2 (1/2000, Novus Biologicals, NB110-57130), anti-TRF1 (1/1000, SantaCruz, sc-56807), anti-TIN2 (1/1000, Sigma-Aldrich, SAB4200108)) diluted in non-fat milk or BSA 5% (w/v) in PBS-T.

Techniques: Fractionation, Isolation, Binding Assay, Control, Immunofluorescence, Proximity Ligation Assay, MANN-WHITNEY, Dot Blot

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet: SKIV2L and TTC37 prevents telomere fragility (A and B) Telomere FISH analysis in SKIV2L- and TTC37-depleted HeLa1.3 and HT1080-ST cells using siRNAs: % of telomere fragility (yellow) and loss (purple) per metaphase in siCtr, siSKIV2L and siTTC37 (means ± SD, n > 25 metaphases, 2 independent experiments, scale bar, 10 μm). t test ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) Telomere FISH analysis in shCtr (Control) or shSKIV2L HEK293 cells expressing GFP or SKIV2L (means ± SD, n > 25 metaphases, 2 independent experiments, scale bar, 10 μm). Details are as in (A). (D) Telomere FISH analysis in TTC37-depleted HEK293 cells using siRNAs (means ± SD, n = 25 metaphases). Details are as in (A). (E) Telomere FISH analysis in HeLa1.3 cells using shRNAs: shCtr and shSKIV2L treated with DMSO (−, control) or APH (+) ( n > 45 metaphases, 2 independent experiments). Details are as in (A). (F) IF of pre-extracted cells showing co-localization of pS1981 ATM autophosphorylation with TRF2 (telomeres) in shCtr and SKIV2L-depleted (shSKIV2L) HeLa1.3 cells. Quantification of the percentage of cells with co-localization foci and mean number of foci per nucleus is depicted (means ± SEM, n = 200 cells, 2 independent experiments, scale bar 15 μm). t test ∗∗∗ p < 0.001. (G) IF-FISH of pre-extracted cells showing co-localization of 53BP1 with telomeres in asynchronous (AS) or G2-synchronized control (shCtr) and SKIV2L-depleted (shSKIV2L) HeLa1.3 cells. Quantification of the percentage of cells with co-localization foci is depicted (means ± SEM, n > 400 cells, 3 independent experiments, scale bar 15 μm). t test ∗ p < 0.05. See also Figure S2 .

Article Snippet: The membrane was blocked using non-fat milk 5% (w/v) in PBS-T (PBS 1X; 0.1% (v/v) Tween-20) and subsequently incubated overnight at 4°C with the primary antibody (anti-RNAseH1 (1/500, Abnova, H00246243-M01), anti-GFP (1/5000, Abcam, ab290), anti-β-actin (1/5000, Abcam, ab8226, used as a loading control), anti-H3 (1/1000, Abcam, ab10799), anti-SKIV2L (1/1000, Proteintech group, 11462-1-AP), anti-TTC37 (1/500, Novus Biologicals, NBP1-93640), anti-WDR61 (1/500, Sigma-Aldrich, SAB1401852; 1/1000, Thermofisher PA5-40079), anti-α-Tubulin (1/1000, Sigma-Aldrich, T6199), anti-TRF2 (1/2000, Novus Biologicals, NB110-57130), anti-TRF1 (1/1000, SantaCruz, sc-56807), anti-TIN2 (1/1000, Sigma-Aldrich, SAB4200108)) diluted in non-fat milk or BSA 5% (w/v) in PBS-T.

Techniques: Control, Expressing

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet: SKIV2L regulates telomeric DNA-RNA hybrids in cellulo to prevent telomere fragility (A) Proximity ligation assay (PLA) showing co-localization of SKIV2L and TRF2 in asynchronous (AS) and in G2-synchronized HeLa1.3 cells with and without RNase H1 (RNH1) overexpression (median, Q1 and Q3, at least 600 cells scored per condition, 4 independent experiments, scale bar 10 μm). Mann-Whitney U test ∗∗∗∗ p < 0.0001. (B) S9.6 IF in HeLa1.3 cells treated with RNAse III, with and without RNase H1 (RNH1) overexpression (median ± interquartile range, 360 cells scored per condition, 4 independent experiments, scale bar, 10 μm). Mann-Whitney U test ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) PLA showing the co-localization of DNA-RNA hybrids (S9.6) and TRF2 in HeLa1.3 cells pre-extracted and treated with RNAse III (median, Q1 and Q3, at least 400 cells scored per condition, 2 independent experiments, scale bar 10 μm). Mann-Whitney U test ∗∗∗∗ p < 0.0001. (D) DRIP showing the levels of DNA-RNA hybrids at telomeres in AS and G2-synchronized HeLa1.3 cells, RNH, RNase H treatment (means ± SEM, n = 4). (E) DRIP-qPCR assay of G2-synchronized HEK293 cells overexpressing GFP or SKIV2L at 10q, 13q, 20q, and 22q subtelomeric regions. RNH, RNase H treatment (means ± SEM, n = 5). Percent input values were normalized to the GFP overexpressing condition. (F) Model proposing the function of hSKI at telomeres. Telomeric DNA-RNA hybrid accumulation in late S/G2 phase drives hSKI recruitment to telomeres to regulate physiological DNA-RNA hybrid levels, prevent telomere replication stress and ensure telomere stability. See also Figure S5 .

Article Snippet: The membrane was blocked using non-fat milk 5% (w/v) in PBS-T (PBS 1X; 0.1% (v/v) Tween-20) and subsequently incubated overnight at 4°C with the primary antibody (anti-RNAseH1 (1/500, Abnova, H00246243-M01), anti-GFP (1/5000, Abcam, ab290), anti-β-actin (1/5000, Abcam, ab8226, used as a loading control), anti-H3 (1/1000, Abcam, ab10799), anti-SKIV2L (1/1000, Proteintech group, 11462-1-AP), anti-TTC37 (1/500, Novus Biologicals, NBP1-93640), anti-WDR61 (1/500, Sigma-Aldrich, SAB1401852; 1/1000, Thermofisher PA5-40079), anti-α-Tubulin (1/1000, Sigma-Aldrich, T6199), anti-TRF2 (1/2000, Novus Biologicals, NB110-57130), anti-TRF1 (1/1000, SantaCruz, sc-56807), anti-TIN2 (1/1000, Sigma-Aldrich, SAB4200108)) diluted in non-fat milk or BSA 5% (w/v) in PBS-T.

Techniques: Proximity Ligation Assay, Over Expression, MANN-WHITNEY

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet:

Article Snippet: The membrane was blocked using non-fat milk 5% (w/v) in PBS-T (PBS 1X; 0.1% (v/v) Tween-20) and subsequently incubated overnight at 4°C with the primary antibody (anti-RNAseH1 (1/500, Abnova, H00246243-M01), anti-GFP (1/5000, Abcam, ab290), anti-β-actin (1/5000, Abcam, ab8226, used as a loading control), anti-H3 (1/1000, Abcam, ab10799), anti-SKIV2L (1/1000, Proteintech group, 11462-1-AP), anti-TTC37 (1/500, Novus Biologicals, NBP1-93640), anti-WDR61 (1/500, Sigma-Aldrich, SAB1401852; 1/1000, Thermofisher PA5-40079), anti-α-Tubulin (1/1000, Sigma-Aldrich, T6199), anti-TRF2 (1/2000, Novus Biologicals, NB110-57130), anti-TRF1 (1/1000, SantaCruz, sc-56807), anti-TIN2 (1/1000, Sigma-Aldrich, SAB4200108)) diluted in non-fat milk or BSA 5% (w/v) in PBS-T.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Blocking Assay, Mass Spectrometry, SYBR Green Assay, Flow Cytometry, Imaging, Mutagenesis, Cell Cycle Assay, shRNA, Software

Journal: Scientific Reports

Article Title: Critical role of SMG7 in activation of the ATR-CHK1 axis in response to genotoxic stress

doi: 10.1038/s41598-021-86957-x

Figure Lengend Snippet: SMG7 maintains normal attenuation of the ATR-CHK1 axis during recovery from replication stress. ( a ) Wild type and SMG7 −/− HCT116 cells were pulsed with 25 μm BrdU followed by treatment with 5 mM HU for 6 h. After HU treatment, cells were released into fresh normal media, and harvested at the indicated time points. Cells were then fixed, stained with α-BrdU antibody (y-axis) and 7-AAD (x-axis) and analyzed by flow cytometry. The BrdU-positive fractions containing G 1 -DNA content from total populations are circled (4, 6 and 8 h after HU removal). ( b – d ) Cells treated as in ( a ) were released into fresh media containing 1 μg/mL nocodazole for different hours. Cells were then fixed and stained with α-BrdU (BU1/75) (green) and α-Histone H3-pS10 (red) antibodies, and imaged using a fluorescence microscope. Representative images of cells 8 h after HU removal are shown in ( b ). Cells were counted using ImageJ, and the percentage of BrdU/H3-pS10 double positive ( c ) cells were quantified. Data are presented as Mean ± SD (n = 3) and analyzed by one-way ANOVA (**** P < 0.0001). ( d ) BrdU-negative/H3-pS10-positive cells were quantified. Data are presented as Mean ± SEM (n = 3) and analyzed by Student’s t-test; P < 0.01. ( e ) Total cell extracts from wild type and SMG7 −/− cells treated as in ( b – d ) were examined by western blot analysis using α-SMG7, α-CHK1-pS345, α-CHK1, α-RPA32-pS33, α-RPA32, α-RAD17 and α-RAD17-pS635 antibodies.

Article Snippet: The following antibodies were from Cell Signaling Technology: α-ATM-pS1981 (5883, 1:1000), α-ATM (2873, 1:1000), α-CHK1-pS345 (2348, 1:1000), α-CHK2-pT68 (2661, 1:1000), α-CHK2 (2662, 1:1000), α-RAD17-pS635 (13404S, 1:1000), α-RAD17 (8561, 1:1000), α-RPA32 (2208S, IF 1:1000), α-Histone H3 (3638, 1:2000), α-rabbit IgG-HRP (7074, 1:5000).

Techniques: Staining, Flow Cytometry, Fluorescence, Microscopy, Western Blot

Journal: Scientific Reports

Article Title: Critical role of SMG7 in activation of the ATR-CHK1 axis in response to genotoxic stress

doi: 10.1038/s41598-021-86957-x

Figure Lengend Snippet: SMG7 promotes chromatin recruitment of RAD9 upon DNA damage. ( a ) Cell extracts from DLD1 SMG7 −/− cells expressing FH-SMG7 and the α-Flag immunoprecipitates were examined by western blot analysis using α-SMG7, α-RAD17, α-ATR, α-TOPBP1, α-CHK1 and α-Tubulin antibodies. ( b , c ) Wild type and SMG7 −/− cells were treated with ionizing radiation (10 Gy, 1 h), pre-extracted, and immunostained in ( b ) with α-RPA32 (green) and α-γH2AX (red) antibodies and in ( c ) with α-RPA32 (green) and α-RAD9 (sc-74464, red). Nuclei were stained with DAPI. Representative images from immunofluorescence microscopy are shown. Wider fields of view at are shown in Fig. S4b-c. ( d ) Quantification of yH2AX-, yH2AX/RPA- and RPA/RAD9- foci-positive cells from experiments represented in ( b , c ). Data are presented as Mean ± SEM (n = 3 independent experiments containing pooled data) and were analyzed by one-way ANOVA with Tukey post-test. **** indicates P < 0.001. ( e ) Wild type and SMG7 −/− DLD1 cells were subjected to fractionation, and the total cells extracts, soluble nuclear and chromatin fractions were analyzed by western blot using α-SMG7 and α-RAD17 antibodies. α-alpha-Tubulin and α-Histone H3 antibodies were used as cytoplasmic and chromatin markers, respectively. Relative levels of chromatin-bound SMG7 and RAD17 are quantitated in Fig. S4d. ( f ) As in ( e ), the chromatin-bound fractions from the control and irradiated cells were isolated and examined by western blot analysis using α-SMG7, α-RAD17, α-RAD17-pS645, α-RAD9 (A300-890A) and α-H3 antibodies. Relative levels of chromatin-bound RAD9 are quantitated in Fig. S4f.

Article Snippet: The following antibodies were from Cell Signaling Technology: α-ATM-pS1981 (5883, 1:1000), α-ATM (2873, 1:1000), α-CHK1-pS345 (2348, 1:1000), α-CHK2-pT68 (2661, 1:1000), α-CHK2 (2662, 1:1000), α-RAD17-pS635 (13404S, 1:1000), α-RAD17 (8561, 1:1000), α-RPA32 (2208S, IF 1:1000), α-Histone H3 (3638, 1:2000), α-rabbit IgG-HRP (7074, 1:5000).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Microscopy, Fractionation, Control, Irradiation, Isolation

Journal: Cell & Bioscience

Article Title: The human testis-specific protein Y-linked (TSPY) is a male-specific cancer-testis antigen capable of eliciting significant immune responses and elimination of positive tumor cells in hepatocellular carcinoma

doi: 10.1186/s13578-025-01432-8

Figure Lengend Snippet: Detection of anti-TSPY antibodies in the sera of positive mice. Protein lysates of 293T cells transfected with either EGFP (293T-EGFP) or TSPY-EGFP (293T-TSPY) and tumors from either Group-1 (G-1) or Group-2 (G-2) mice were analyzed with western blots using sera from Group-1 and Group-2 mice. The serum of individual Group-1 mice detected only the EGFP protein in 293T cells transfected with EGFP or TSPY-EGFP vector while those from Group-2 mice detected TSPY protein only in 293T cells transfected with TSPY-EGFP construct or Group-2 mice harboring TSPY-positive tumors. Left 2 panels (293T cells), right 4 panels, mouse tumor protein lysates. A rabbit polyclonal antibody against TSPY (Anti-TSPY rabbit poly) and a monoclonal antibody against the FLAG epitope (Anti-FLAG mouse mono) were used as references for detection of various fragments and full length TSPY protein respectively. A goat anti-GFP polyclonal antibody (Anti-GFP goat poly) was used to detect the EGFP protein in the tumors (rightmost panel). Detection of various TSPY fragments in both transfected 293T cells and positive tumors suggested that the TSPY protein was degraded/processed by proteasomal mechanisms in both cultured 293T cells and tumors of positive mice

Article Snippet: Western blotting of protein lysates (from cell cultures and mouse tissues) were performed as previously described [ ], using anti-TSPY rabbit antibody (1:4000 dilution, in house) [ ], anti-B2M rabbit polyclonal antibody (1:2000 dilution, Proteintech Group), anti-βactin mouse monoclonal antibody (clone AC-15, 1:8000 dilution, Sigma-Aldrich), anti-GFP goat polyclonal antibody (1:2000 dilution, Abcam, Waltham, MA), or serum from Group-1 or Group-2 mice (1:2000 dilution).

Techniques: Transfection, Western Blot, Plasmid Preparation, Construct, FLAG-tag, Cell Culture

Journal: Cell & Bioscience

Article Title: The human testis-specific protein Y-linked (TSPY) is a male-specific cancer-testis antigen capable of eliciting significant immune responses and elimination of positive tumor cells in hepatocellular carcinoma

doi: 10.1186/s13578-025-01432-8

Figure Lengend Snippet: Demonstration of TSPY protein location on cell surface using flow cytometry and cell fractionation analyses on HCC HuH7 tumor cells. HuH7 cells were transfected with either an EGFP or a TSPY-EGFP vector, representing TSPY negative and positive cells respectively. ( A ) They were analyzed with flow cytometry for cell surface proteins using a panel of 9 TSPY monoclonal antibodies (5 presented here) showing various degrees of bindings to the surface of TSPY-positive cells (pink) and shifting to the right from the negative cells (blue). A rabbit TSPY polyclonal antibody was used as a reference (bottom right). ( B ) Diagrammatic illustration on the approximate locations of the epitopes of respective monoclonal antibodies along the TSPY protein. ( C ) HuH7 cells negative (blue label) and positive (red label) for TSPY were fractionated into intracellular and cell surface components and analyzed with western blotting with antibodies against TSPY (top row), anti-βactin (an intracellular marker, middle row) and anti-β2-microglobulin (B2M a cell surface marker, bottom row), showing the presence of TSPY protein in both intracellular and cell surface fractions corresponding to the respective intracellular and cell surface markers. Bottom right showed similar western blot analysis with Group-2 mouse sera. Red arrows indicate the respective TSPY band, despite in small quantity, on the cell surface fraction

Article Snippet: Western blotting of protein lysates (from cell cultures and mouse tissues) were performed as previously described [ ], using anti-TSPY rabbit antibody (1:4000 dilution, in house) [ ], anti-B2M rabbit polyclonal antibody (1:2000 dilution, Proteintech Group), anti-βactin mouse monoclonal antibody (clone AC-15, 1:8000 dilution, Sigma-Aldrich), anti-GFP goat polyclonal antibody (1:2000 dilution, Abcam, Waltham, MA), or serum from Group-1 or Group-2 mice (1:2000 dilution).

Techniques: Flow Cytometry, Cell Fractionation, Transfection, Plasmid Preparation, Bioprocessing, Western Blot, Marker

Journal: Cell & Bioscience

Article Title: The human testis-specific protein Y-linked (TSPY) is a male-specific cancer-testis antigen capable of eliciting significant immune responses and elimination of positive tumor cells in hepatocellular carcinoma

doi: 10.1186/s13578-025-01432-8

Figure Lengend Snippet: Involvement of MHC-I complex in the cellular immune elimination of tumor cells. The MHC-I complexes are important for presentation of self and non-self entities of cellular proteins on the surface and responsible for cellular immune responses to antigenic cancer-testis antigen, i.e. TSPY. Using a CRISPR strategy to knockout a key component, i.e. β2-microglobulin (B2M), of the MHC-I complex, such peptide-MHC-I complex formation and cellular immune responses could be minimized/eliminated. Tumors derived from AKT + NRAS + TSPY-EGFP tumors co-injected with a non-specific (control) sgRNA in the CRISPR procedure showed only small tumor foci ( A & B ), which were positive for both TSPY and B2M protein ( E & F ), suggesting they could be subjected to cellular immune responses and elimination. Those injected with specific sgRNA ( C & D ) showed two types of large tumor foci in addition to the small foci. One minor type of large tumor foci was negative for both TSPY and the co-expressed EGFP ( C & D , yellow arrowheads), representing no TSPY expression in the tumor cells. The others represented the major type of large tumor foci (white arrows), showing positive for TSPY and the co-expressed EGFP ( D & G ) but negative for B2M ( H ). Thus, these large tumor foci escaped immune elimination by cytotoxic T cells through either deleting/suppressing the antigenic TSPY protein expression or minimizing TSPY peptide-HMC-I complex formation and cell surface presentation in the absence of b2-microglobulin (B2M). Scale bar = 1 cm in A-D; 200 μm in E-H

Article Snippet: Western blotting of protein lysates (from cell cultures and mouse tissues) were performed as previously described [ ], using anti-TSPY rabbit antibody (1:4000 dilution, in house) [ ], anti-B2M rabbit polyclonal antibody (1:2000 dilution, Proteintech Group), anti-βactin mouse monoclonal antibody (clone AC-15, 1:8000 dilution, Sigma-Aldrich), anti-GFP goat polyclonal antibody (1:2000 dilution, Abcam, Waltham, MA), or serum from Group-1 or Group-2 mice (1:2000 dilution).

Techniques: CRISPR, Knock-Out, Derivative Assay, Injection, Control, Expressing

Journal: Pharmaceutical biology

Article Title: Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation.

doi: 10.1080/13880209.2023.2173248

Figure Lengend Snippet: Figure 7. PKM2 nuclear accumulation promoted phospho-STAT3 at Y705 in Th17 cells. (A) The mRNA expression of STAT3 in CD4þT cells (with the condition of Th17- polarization) treated with Tepp-46 (50, 100, and 150 lM) was evaluated by RT-qPCR. (B,C) Tepp-46 (50, 100 and 150lM)-treated Th17 cell lysates were subjected to western blot of total and phospho-STAT3 (Y705) expression. b-Actin was used as a loading control. (D) The mRNA expression of STAT3 in CD4þT cells (with the condi- tion of Th17-polarization) treated with SAICAR (2, 4 and 8 lM) was evaluated by RT-qPCR. (E,F) SAICAR (2, 4 and 8 lM)-treated Th17 cell lysates were subjected to western blot of total and phospho-STAT3 (Y705) expression. b-Actin was used as a loading control. Data were expressed as the mean ± SEM (n ¼ 3). p < 0.05, p < 0.01, p < 0.001 compared with the control group by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA), incubated with PKM2 primary antibody (Proteintech, 15822-1-AP; 1:1000 dilution) or STAT3 primary antibody (CST, D3Z2G; 1:1000 dilution) or phosphor-STAT3 (Tyr705) (CST, D3A7; 1:2000 dilution) overnight at 4 C and secondary HRP-conjugated antibodies (proteintech, SA00001-2; 1:3000 dilution) for 1.5 h. Images were obtained and analyzed with the Image Lab software (Bio-Rad).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Pharmaceutical biology

Article Title: Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation.

doi: 10.1080/13880209.2023.2173248

Figure Lengend Snippet: Figure 8. PNS blocked STAT3 phosphorylation and inhibited Th17 cell differentiation by suppressing PKM2 nuclear accumulation. Naïve CD4þT cells (with the condi- tion of Th17-polarization) were pretreated with SAICAR (4 lM) or Tepp-46 (100lM) for 24 h, and treated with PNS (10lg/mL) for another 72 h. (A,B) Nuclear and cyto- plasmic fractions were isolated by cell fractionation and analyzed for PKM2 expression by western blots. Histone H3 and b-actin were used as nuclear and cytoplasm loading controls, respectively. (C,D) The Th17 cell lysates were subjected to western blot of total and phospho-STAT3 (Y705) expression. b-Actin was used as a loading control. (E,F) The mRNA relative expression of RORct was detected by RT-qPCR. (G,H) Representative dot plots and the percentage of Th17 cells (CD4þIL-17þ cells) were determined by flow cytometry. Data were expressed as the mean ± SEM (n ¼ 3). p < 0.05 compared with the control group, #p < 0.05, ##p < 0.01 compared with the SAICAR or Tepp-46 group by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA), incubated with PKM2 primary antibody (Proteintech, 15822-1-AP; 1:1000 dilution) or STAT3 primary antibody (CST, D3Z2G; 1:1000 dilution) or phosphor-STAT3 (Tyr705) (CST, D3A7; 1:2000 dilution) overnight at 4 C and secondary HRP-conjugated antibodies (proteintech, SA00001-2; 1:3000 dilution) for 1.5 h. Images were obtained and analyzed with the Image Lab software (Bio-Rad).

Techniques: Phospho-proteomics, Cell Differentiation, Isolation, Cell Fractionation, Expressing, Western Blot, Control, Quantitative RT-PCR, Flow Cytometry

Journal: Pharmaceutical biology

Article Title: Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation.

doi: 10.1080/13880209.2023.2173248

Figure Lengend Snippet: Figure 9. PNS repressed the PKM2/STAT3 signaling pathway to attenuate inflammation in the CIA model. DBA/1J mice were immunized with bovine type II collagen emulsified in complete Freund adjuvant and incomplete Freund adjuvant. Mice were treated at day 21 after the first immunization with saline or PNS (100mg/kg). (A) Nuclear and cytoplasmic fractions were isolated by cell fractionation from splenic CD4þT cells of CIA mice and analyzed for PKM2 expression by western blots. Histone H3 and b-actin were used as nuclear and cytoplasm loading controls, respectively. (B) Splenic CD4þT cells from CIA mice were crosslinked with DSS and ana- lyzed for dimeric/tetrameric PKM2 expression by western blots. b-Actin was used as a loading control. (C) Splenic CD4þT cell lysates from CIA mice were subjected to a western blot of total and phospho-STAT3 (Y705) expression. b-Actin was used as a loading control. Data were expressed as the mean ± SEM (n ¼ 6). p < 0.05, p < 0.01, compared with the control group, #p < 0.05, ##p < 0.01, compared with the model group, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA), incubated with PKM2 primary antibody (Proteintech, 15822-1-AP; 1:1000 dilution) or STAT3 primary antibody (CST, D3Z2G; 1:1000 dilution) or phosphor-STAT3 (Tyr705) (CST, D3A7; 1:2000 dilution) overnight at 4 C and secondary HRP-conjugated antibodies (proteintech, SA00001-2; 1:3000 dilution) for 1.5 h. Images were obtained and analyzed with the Image Lab software (Bio-Rad).

Techniques: Adjuvant, Saline, Isolation, Cell Fractionation, Expressing, Western Blot, Control

Journal: Pharmaceutical biology

Article Title: Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation.

doi: 10.1080/13880209.2023.2173248

Figure Lengend Snippet: Figure 10. PNS attenuates Th17 cell differentiation via inhibition of nuclear PKM2-mediated STAT3 phosphorylation. The cooperation between TCR activation and cos- timulatory signals leads to a significant increase of PKM2 expression. IL-6 and IL-23 are the important cytokines for controlling the Th17 cell differentiation, IL-6R and IL-23R signaling cascade promote STAT3 phosphorylation/activation, companying with an accumulation of dimeric PKM2 in Th17 cells. The dimeric oligomer state facil- itates PKM2 translocation into the nucleus and enhancing STAT3 phosphorylation, contributing to increase its transcriptional activity. This process ultimately enhances the transcription of Th17 cell–associated genes. PNS specifically inhibit PKM2 dimerization and nuclear accumulation, and further induced the decrease of STAT3 phos- phorylation, contributing to suppress Th17 cell differentiation.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA), incubated with PKM2 primary antibody (Proteintech, 15822-1-AP; 1:1000 dilution) or STAT3 primary antibody (CST, D3Z2G; 1:1000 dilution) or phosphor-STAT3 (Tyr705) (CST, D3A7; 1:2000 dilution) overnight at 4 C and secondary HRP-conjugated antibodies (proteintech, SA00001-2; 1:3000 dilution) for 1.5 h. Images were obtained and analyzed with the Image Lab software (Bio-Rad).

Techniques: Cell Differentiation, Inhibition, Phospho-proteomics, Activation Assay, Expressing, Translocation Assay, Activity Assay

Journal: bioRxiv

Article Title: CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation

doi: 10.1101/2024.10.19.619045

Figure Lengend Snippet: A - Illustration of modified transposase donor plasmids to test the effect of alternative NLSs on dCas9-KRAB localization and stability in neurons (drawn to scale). B - Flow cytometry plots measuring expression of modified dCas9-KRAB constructs or nCas9 via tagBFP fluorescence in neurons 14 days post induction of differentiation with dox from stably integrated iPSCs. Red line indicates the approximate cutoff for positive cells based on parental line autofluorescence. Plots represent a minimum of 3000 analyzed single cells. C - Quantification of flow cytometry data showing the mean fluorescence intensity of tagBFP normalized to autofluorescence of dCas9-KRAB constructs and nCas9 in Day 14 neurons (Parental autofluorescence=1). Alternative NLS sequences rescue dCas9-KRAB expression to levels comparable to nCas9, with the 2xMeCP2 NLS version having the highest expression with a mean population intensity of 4.931x the parental, compared to a mean of 4.586x for the MeCP2 NLS construct and 4.636x for the cMyc NLS+MeCP2 NLS construct. Data represent the mean fluorescence intensity of 2-3 wells per line with a minimum of 2000 analyzed single cells per sample. Error bars represent mean ± SD. D - Representative Western blots (left) and quantification (right) of total protein from Day 14 neurons probed for Cas9 showing significant rescue of total protein levels of 2xMeCP2 NLS-dCas9-KRAB compared to the original dCas9-KRAB, with levels comparable to nCas9. Cas9 antibody signal was normalized to TUBB3 levels. Data represents 3 independent wells per line. Error bars represent mean ± SD. Protein levels between lines were compared with One-way ANOVA with Tukey’s multiple comparisons test. ** P=0.0050 for dCas9-KRAB vs Cas9, * P=0.0150 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, ns P=0.5719 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. E-Representative Western blots (left) and quantifications (right) of dCas9-KRAB or nCas9 protein levels in nuclear and cytoplasmic protein fractions from Day 14 neurons. The original dCas9-KRAB and nCas9 are both predominantly cytoplasmic with nearly identical distributions despite much lower dCas9-KRAB levels. The 2xMeCP2 NLS-dCas9-KRAB construct shows a significant shift into the nuclear fraction which corresponds with the rescue of total protein levels. LaminB1 and GAPDH were used as nuclear and cytoplasmic controls, respectively, and were probed on replicate sample blots in parallel with Cas9. Quantifications for each protein represent the intensity of the signal in the respective fraction divided by the sum of intensities across both fractions. Cas9 quantifications were performed on respective LaminB1 and GAPDH blots. Data represent 3 independent wells per line. Error bars represent mean ± SD. Fractionation control and Cas9 antibody signals across cell lines were compared with 2-way ANOVA with Tukey’s multiple comparisons test. For Cas9 on LaminB1 blots: ns P=0.1236 for dCas9-KRAB vs nCas9, **** P<0.0001 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, **** P<0.0001 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. For Cas9 on GAPDH blots: ns P=0.6805 for dCas9-KRAB vs nCas9, **** P<0.0001 for dCas9-KRAB vs 2xMeCP2 NLS-dCas9-KRAB, **** P<0.0001 for nCas9 vs 2xMeCP2 NLS-dCas9-KRAB. F - Representative images of dCas9-KRAB protein localization in Day 14 neurons with ICC using a Cas9 antibody (green). Cells are stained with a MAP2 antibody (red) and nuclei with NucSpot 750/780 (magenta). Nuclei are outlined for reference. 2xMeCP2 NLS-dCas9-KRAB shows improved nuclear localization over original nCas9 and dCas9-KRAB constructs and higher intensity staining than the original dCas9-KRAB, consistent with the results from biochemistry assays. Images are Max IPs of 4-plane Z-stacks (0.6um) taken at 40x with a spinning disk confocal. All images are adjusted to the same LUTS, based on background Cas9 antibody staining in the Parental line. Scale bars represent 20um. G - Quantification of RT-qPCR data showing significantly improved knockdown of PSAP (top) and SNCA (bottom) in neurons expressing the 2xMeCP2 NLS-dCas9-KRAB vs the original dCas9-KRAB. Neurons were transduced with lentivirus expressing an mScarlet marker with either a targeting or non-targeting sgRNA at Day 14 (PSAP) or Day 21 (SNCA) post-differentiation and RNA harvested 7-days post-transduction. Gene expression levels were normalized to GAPDH and ACTB and are shown relative to the non-targeting average. Data represent 3 independent wells per transduction per line. 2-way ANOVA with uncorrected Fisher’s LSD was used to compare targeting guide with non-targeting guide within cell lines and targeting guides across cell lines. For PSAP KDs: ns P=0.0745 for dCas9-KRAB+NTCg vs +PSAPg, *** P=0.0004 for 2xMeCP2 NLS-dCas9-KRAB+NTCg vs +PSAPg, ** P=0.006 for dCas9-KRAB+PSAPg vs 2xMeCP2 NLS-dCas9-KRAB+PSAPg. For SNCA KDs: * P=0.0152 for dCas9-KRAB+NTCg vs +SNCAg, **** P<0.0001 for 2xMeCP2 NLS-dCas9-KRAB+NTCg vs +SNCAg, ** P=0.0035 for dCas9-KRAB+SNCAg vs 2xMeCP2 NLS-dCas9-KRAB+SNCAg.

Article Snippet: The following primary antibodies were used: Cas9 (Cell Signaling, 14697; 1:1000), TUBB3 (Abcam, ab18207; 1:800), LaminB1 (Proteintech, 12987-1-AP; 1:2000), GAPDH (Cell Signaling, 2118; 1:500).

Techniques: Modification, Flow Cytometry, Expressing, Construct, Fluorescence, Stable Transfection, Western Blot, Fractionation, Control, Staining, Quantitative RT-PCR, Knockdown, Transduction, Marker