Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: TUG1 promotes AGE1.HN and PC12 cell death. (a) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells treated with and without hypoxia measured by western blot analysis normalized to β-actin (unpaired t test, ***p < 0.001. For c-caspase3: AGE1.HN: t(5.061) = −35.105, p < 0.001; PC12: t(8) = −25.322, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: t(8) = −49.07, p < 0.001; PC12: t(8) = −18.78, p < 0.001). Human or rat pcDNA-TUG1 and siRNA-TUG1 and their NCs were transfected into hypoxia-treated AGE1.HN and PC12 cells, respectively. (b) TUG1 expression in AGE1.HN and PC12 cells after transfection measured by RT-qPCR normalized to GAPDH (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 35,058.432, p < 0.001; PC12: F(3,16) = 70,046.402, p < 0.001). (c) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells after transfection measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.001. For c-caspase3: AGE1.HN: F(3,16) = 416.825, p < 0.001; PC12: F(3,16) = 260.449, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: F(3,16) = 475.2, p < 0.001; PC12: F(3,16) = 468.6, p < 0.001). (d) the death rate of AGE1.HN and PC12 cells after transfection measured by flow cytometry (one-way ANOVA, *p = 0.019, **p = 0.005, ***p < 0.001. AGE1.HN: F(3,16) = 184.670, p < 0.001; PC12: F(3,16) = 34.701, p < 0.001). (e) the distribution of TUG1 in AGE1.HN and PC12 cells evaluated by nuclear/cytoplasmic fractionation. Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Fractionation, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: BIK facilitates AGE1.HN and PC12 cell death. AGE1.HN and PC12 cells were delivered with pcDNA-BIK or siRNA-BIK. (a) the BIK mRNA expression in AGE1.HN and PC-12 cells determined by RT-qPCR (one-way ANOVA, ***p < 0.001. AGE1.HN: F(3,16) = 9423.939, p < 0.001; PC12: F(3,16) = 19,510.157, p < 0.001). (b) the protein expression of c-caspase3 and c-caspase3/total caspase3 in AGE1.HN and PC12 cells after transfection measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.01. For c-caspase3: AGE1.HN: F(3,16) = 750.932, p < 0.001; PC12: F(3,16) = 1644.487, p < 0.001. For c-caspase3/total caspase3: AGE1.HN: F(3,16) = 583.2, p < 0.001; PC12: F(3,16) = 513.6, p < 0.001). (c) the death rate of AGE1.HN and PC12 cells after transfection measured by flow cytometry (one-way ANOVA, **p = 0.002, ***p < 0.001. AGE1.HN: F(3,16) = 648.677, p < 0.001; PC12: F(3,16) = 517.799, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Flow Cytometry, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: The regulatory role of TUG1/miR-338/BIK axis on rats with SCI (n = 5). (a) the BBB scores for hindlimb locomotion in the rats with ASCI injected with siRNA-TUG1 alone or with miR-338 inhibitor (one-way ANOVA, **p = 0.008, ***p < 0.001. F(3,16) = 175.553, p < 0.001). (b) detection of cell death in rat spinal cord by TUNEL staining (one-way ANOVA, ***p < 0.001. F(5,24) = 296.154, p < 0.001). (c) TUG1 (normalized to GAPDH) and miR-338 expression (normalized to U6) as well as the BIK mRNA expression (normalized to GAPDH) in spinal cord tissues of rats determined by RT-qPCR (one-way ANOVA, ***p < 0.001. TUG1: F(3,16) = 140.714, p < 0.001; miR-338: F(3,16) = 291.155, p < 0.001; BIK: F(3,16) = 251.190, p < 0.001). (d) the protein expression of c-caspase3 and c-caspase3/total caspase3 in spinal cord tissues of rats measured by western blot analysis normalized to β-actin (one-way ANOVA, ***p < 0.001. For c-caspase3/total caspase3: F(3,16) = 146.102, p < 0.001. For c-caspase3/total caspase3: F(3,16) = 702.9, p < 0.001). Data were displayed as the mean ± SD. Five independent assays were conducted only for in vitro experiments

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Injection, TUNEL Assay, Staining, Expressing, Quantitative RT-PCR, Western Blot, In Vitro

Journal: Bioengineered

Article Title: Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

doi: 10.1080/21655979.2021.1966258

Figure Lengend Snippet: Mechanisms of TUG1 mediated SCI progression. TUG1 interacts with miR-338, leading to BIK activation, which accelerates SCI via promoting cell death. While TUG1 knockdown downregulates BIK expression by interacting with miR-338, thus lowering c-caspase3 to hamper death

Article Snippet: The membranes were sealed for 1 h with a tris buffered saline with Tween-5% skim milk at room temperature and probed with 5% bovine serum albumin-diluted primary antibodies to HIF-1α (1:1000, ab179483, Abcam, Cambridge, UK), β-actin (1:2500, ab8227, Abcam), total caspase3 (1:2000, ab184787, Abcam), and c-caspase3 (1:5000, #9664, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C.

Techniques: Activation Assay, Expressing

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