non targeting scrambled sirna  (Keygen Biotech)

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B drives GC cell invasion and migration in a PI3K/AKT-dependent manner. (A) Gene set enrichment analysis indicated that genes upregulated in the UPK1B-high group were enriched in the PI3K/AKT pathway. (B) Protein levels of UPK1B in GC cell lines. (C) Knockdown of UPK1B reduced PI3K/AKT activation in MKN45 cells. Silencing UPK1B suppressed the (D) migration/invasion capacity and (E) wound closure rate of MKN45 cells. (F) Overexpression of UPK1B enhanced PI3K/AKT pathway activation in AGS cells, which was attenuated by the PI3K inhibitor LY294002. Inhibition of PI3K/AKT signaling reversed UPK1B-induced (G) migration/invasion capacity and (H) wound closure rate of AGS cells. UPK1B, uroplakin 1B; GC, gastric cancer; p-, phosphorylated; sh, short hairpin RNA; NC, negative control; OE, overexpression.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Migration, Knockdown, Activation Assay, Over Expression, Inhibition, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: CDX2 acts as a transcriptional repressor of UPK1B and its high expression is associated with favorable prognosis of patients with GC. (A) Venn diagram showing overlapping predicted transcriptional regulators of UPK1B from ChEA and ChEA3 databases. (B) Knockdown of CDX2 in AGS cells resulted in increased UPK1B (C) mRNA and (D) protein expression. (E) Overexpression of CDX2 in MKN45 cells reduced UPK1B protein levels. Data from (F) The Cancer Genome Atlas Stomach Adenocarcinoma cohort and (G) the Kaplan-Meier plotter database indicated that high CDX2 expression was associated with improved prognosis of patients with GC. UPK1B, uroplakin 1B; GC, gastric cancer; si, small interfering RNA; NC, negative control; OE, overexpression; HR, hazard ratio; CDX2, caudal-related homeobox transcription factor 2; ChEA, ChIP-X Enrichment Analysis.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Expressing, Knockdown, Over Expression, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: CDX2-UPK1B-PIK3IP1-PI3K/AKT signaling axis regulates gastric cancer cell invasion and migration and influences patient prognosis

doi: 10.3892/etm.2026.13179

Figure Lengend Snippet: UPK1B activates PI3K/AKT signaling by antagonizing the inhibitory regulator PIK3IP1 in gastric cancer cells. (A) Venn diagram showing that PIK3IP1 was identified as a putative UPK1B-interacting partner based on BioGRID and HIPPIE protein-protein interaction databases. (B) UPK1B and PIK3IP1 co-localized in the cytoplasm and plasma membrane of MKN45 cells. (C) Interaction between UPK1B and PIK3IP1 in MKN45 cells. (D) Knockdown of PIK3IP1 in MKN45 cells. (E) Silencing PIK3IP1 in UPK1B-knockdown MKN45 cells restored PI3K/AKT pathway activation. Knockdown of PIK3IP1 reversed the decrease in (F) migration/invasion and (G) wound-healing capacity in UPK1B-silenced MKN45 cells. UPK1B, uroplakin 1B; p-, phosphorylated; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; PIK3IP1, PI3K inhibitor interacting protein 1; HIPPIE, Human Integrated Protein-Protein Interaction Reference; IP, immunoprecipitation.

Article Snippet: Cells were also transfected with small interfering RNAs (siRNAs) targeting CDX2 or PIK3IP1 , with a universal non-targeting scrambled siRNA (si-NC) as the negative control , obtained from GeneChem, Inc. For UPK1B and CDX2 overexpression, the p-TSB-CMV-UPK1B and p-TSB-CMV-CDX2 expression vectors [Shanghai Genomeditech Co., Ltd.] and the corresponding empty p-TSB-CMV vector (negative control) were used.

Techniques: Clinical Proteomics, Membrane, Knockdown, Activation Assay, Migration, Small Interfering RNA, shRNA, Negative Control, Immunoprecipitation

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) Western blot analysis showing expression of CHOP in control, S1P, and GSK pretreated S1P-treated CD8 + T cells. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( n = 3). ( B ) q-PCR analysis of Ddit3 (encoding CHOP) in respective groups ( n = 3). ( C , D ) Western blot analysis showing expression of CHOP in activated CD8 + T cells upon S1pr1 knockdown using ( C ) siRNA and ( D ) shRNA. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( N = 3, for both ( C , D ). ( E ) Purified mouse CD8⁺ T cells activated under the indicated treatment conditions were analyzed for the production of effector cytokines. The adjacent bar plots represent cumulative data from three biological replicates ( n = 3). ( F ) Purified mouse CD8⁺ T cells activated under the indicated treatment conditions were assessed for the frequency of CD8 + T cells undergoing apoptosis, as determined by Annexin V and 7AAD staining. The adjacent bar plots represent cumulative data from four biological replicates ( n = 4). ( G – L ) C57BL/6 mice ( n = 4 mice/group) with subcutaneously established YUMM1.7 melanoma tumor treated either with vehicle control or GSK, as ( G ) represented schematically, were evaluated for: ( H ) tumor growth, ( I ) the ability of CD8 + T cells from the tumor site to produce different effector cytokines, ( J ) frequency of CD8 + T cells at the tumor site, ( K ) expression of PD1, and ( L ) expression of Tim3 on intratumoral CD8 + T cells. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( I – L ), one-way ANOVA ( A – F ), and two-way ANOVA test ( H ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Western Blot, Expressing, Control, Knockdown, shRNA, Purification, Staining, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) Western blot analysis of phospho-p38 (p-p38) and total p38 expression, in vehicle control and S1P-treated CD8 + T cells. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( n = 3). ( B , C ) Western blot analysis showing the expression of p-p38 and total p38 in activated T cells upon S1pr1 knockdown using ( B ) siRNA ( n = 3) and (C) shRNA ( n = 3). The adjacent bar graph depicts normalized densitometric data. ( D ) Western blot analysis of p-p38 and total p38 in CD8 + T cells activated in the presence or absence of S1P, along with the indicated inhibitor. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( n = 3). ( E , F ) qPCR analysis of transcript levels of different ( E ) Map3k and (F) Map2k genes in CD8 + T cells in respective groups ( n = 4). ( G ) CD8 + T cells were activated in the presence or absence of S1P and were collected and processed for chromatin-immunoprecipitation (ChIP) assay with an antibody specific for CHOP or with rabbit IgG control. qPCR primers specific for the known CHOP binding gene ( Dr5 ) and different Map3K and Map2K , along with Mapk14 , were used to determine CHOP binding to the respective promoters ( n = 4). ( H , I ) Purified mouse CD8⁺ T cells activated under the indicated treatment conditions were assessed for: ( H ) T cell death by Annexin V and 7AAD staining and ( I ) frequency of CD8 + T cells producing different effector cytokines. The adjacent bar represents cumulative data from four biological replicates ( n = 4, for both ( H , I )). ( J ) Extracellular flux assay for determining of oxygen consumption rate (OCR) in activated CD8 + T cells in respective groups. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A ), one-way ANOVA ( B – D , H , I ), and two-way ANOVA test ( E – G ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Western Blot, Expressing, Control, Knockdown, shRNA, Chromatin Immunoprecipitation, Binding Assay, Purification, Staining, XF Assay, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) CD8⁺ T cells isolated from either the tumor site or spleen of C57BL/6 mice ( n = 4) bearing YUMM1.7 melanoma were assessed for p-p38 expression. The adjacent bar plot summarizes pooled data from four tumor-bearing mice. ( B ) Intratumoral CD8⁺ T cells from C57BL/6 mice ( n = 4/group) with subcutaneous YUMM1.7 melanoma, treated with vehicle control or p38i, were evaluated for the frequency of terminally exhausted CD8⁺ T cells (PD1⁺Tim3⁺). The adjacent bar plot summarizes pooled data from four mice per group. ( C ) Adoptively transferred Pmel-1 T cells transduced with either control shRNA or shRNA targeting PERK, isolated from tumors of C57BL/6 mice ( n = 4/group) bearing subcutaneous B16-F10 melanoma and treated with or without anti-PD1 antibody, were evaluated for the frequency of terminally exhausted CD8⁺ T cells (PD1⁺Tim3⁺). The adjacent bar plot summarizes pooled data from four mice per group. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001 ns, nonsignificant ( P > 0.05). The error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A , B ) and one-way ANOVA. .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Isolation, Expressing, Control, Transduction, shRNA, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A – D ) C57BL/6 mice ( n = 4 mice/group) with subcutaneously established YUMM1.7 melanoma tumor treated either with vehicle control or p38i, as ( A ) represented schematically, were evaluated for: ( B ) tumor growth, ( C ) frequency of CD8 + T cells at the tumor site, ( D ) the ability of CD8 + T cells from the tumor site to produce different effector cytokines. ( E ) Schematic representation of the ACT protocol where C57BL/6 mice ( n = 4 mice/group) bearing subcutaneous B16-F10 tumors were adoptively transferred with 0.75 × 10⁶ Pmel-1 T cells, followed by treatment with or without anti-PD1 antibody (Clone# RMP1-14; 200 µg/mouse twice weekly), combined with p38i or vehicle control. Mice were subsequently evaluated for: ( F ) tumor growth ( n = 4), ( G ) frequency of Vβ13 + CD8 + T cells at the tumor site ( n = 4), and ( H ) Intracellular expression of effector cytokines in intratumoral Pmel-1 T cells following in vitro restimulation ( n = 4). ( I ) Schematic representation of the ACT protocol where C57BL/6 mice ( n = 4 mice/group) bearing subcutaneous B16-F10 tumors were adoptively transferred with 0.75 × 10⁶ Pmel-1 T cells transduced with either control shRNA (Pmel WT ) or shRNA targeting PERK (Pmel PERK ), followed by treatment with or without anti-PD1 antibody (Clone# RMP1-14; 200 µg/mouse twice weekly). Mice were evaluated for: ( J ) tumor growth ( n = 4), ( K ) frequency of Vβ13 + CD8 + T cells at the tumor site ( n = 4), and ( L ) Intracellular expression of effector cytokines in intratumoral Pmel-1 T cells following in vitro restimulation ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( C , D ), one-way ANOVA ( G , H , K , L ), and two-way ANOVA test ( B , F , J ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Control, Expressing, In Vitro, Transduction, shRNA, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Inhibiting HSP27 activates the XBP1s/CerS1 interplay, which triggers DRP1-driven mitophagy, thereby protecting against cell death and promoting the KSHV lytic cycle in primary effusion lymphoma cells

doi: 10.1038/s41420-026-02979-2

Figure Lengend Snippet: BC3 and BCBL1 cell lines were treated or not with 10 and 20 μM J2 (HSP27 inhibitor). After 24 h ( A ) the cytotoxic effect of J2 was measured by trypan blue assay. The histograms represent the percentage of cell viability relative to the control. Data are shown as the mean plus S.D. of three experiments. p value * <0.05, as calculated by ANOVA test. B BiP and CHOP expression as evaluated by western blot analysis. Histone H3 (H3) was used as loading control. The histograms represent the densitometric analysis of the ratio of BiP/H3 and CHOP/H3. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test. BC3 and BCBL1 cell lines treated or not with J2 (20 μM) for 4 h were analyzed by western blot for the expression of XBP1s ( C ) and after 24 h of treatment for cleaved PARP (clPARP) ( D ). GAPDH was used as loading control. The histograms represent the densitometric analysis of the ratio of XBP1s /GAPDH and clPARP/GAPDH. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by Student’s t -test. BC3 and BCBL1 cell lines were knocked down for HSP27 (siHSP27) or scramble treated, using siRNA from different companies (Santa Cruz and Dharmacon respectively), for 24 h and the expression of BiP ( E ) and XBP1s was evaluated by western blot analysis while the effect on cell viability was evaluated by trypan blue assay ( F ). Histone H3 (H3) was used as loading control. The histograms represent the densitometric analysis of the ratio of HSP27/H3, BiP/H3 and XBP1s/H3. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by Student’s t -test.

Article Snippet: For transfection 10 μl of INTERFERin reagent (Polyplus, Illkirch, France; cat. n. 101000016) in combination with 50 pmol of specific small interfering RNA (siRNA duplex) or with non-targeting (scramble) siRNA (Santa Cruz Biotechnology, Inc., Heidelberg, Germany cat. n. sc-29350) were diluted in 200 μl RPMI without serum and antibiotics, incubated for 10 minutes at RT then added to each well.

Techniques: Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: Neurogranin enhances spontaneous activity and neuronal survival of hippocampal neurons

doi: 10.64898/2026.01.27.701932

Figure Lengend Snippet: Primary hippocampal neurons were infected with AAV-Ng at DIV7 or with AAV-shNg or scrambled shRNA (AAV-shScr) at DIV10 and analyzed at DIV15. (a) Ng protein levels were assessed by Western blot and normalized to GAPDH (upper panel; n = 4). The proportion of Ng-positive neurons was determined by fundingimmunofluorescence using DAPI to identify total cells (lower panel; n = 4). (b) Dendritic length per neuron, quantified for each field of view, increased following Ng expression. (c) Synaptic density was analyzed by co-localization of excitatory (vGluT1/PSD95) and inhibitory (GAD65/Gephyrin) pre- and postsynaptic markers. Data in histograms are total number of synapses per field of view (0.11 mm 2 ). Ng expression increased the number of excitatory and inhibitory synaptic contacts (yellow puncta; n = 52–58 images). (d) The axon initial segment (AIS) was labeled with anti-ankyrin G (AnkG). Total AIS length (left graph; n = 25 neurons per condition) and distances from soma to AIS onset, peak intensity, and distal end (right graph) were quantified using DAPI to locate the soma. Widefield images (a–c) were acquired on a Zeiss Axiovert 200M microscope with 10× (A, B) or 40× (c) objectives; AIS images (d) were obtained using a Zeiss LSM900 confocal microscope with a 63× objective.

Article Snippet: As a control, a non-targeting scrambled shRNA (gtgccaagacgggtagtca, Addgene #181875) was used.

Techniques: Infection, shRNA, Western Blot, Expressing, Labeling, Microscopy