Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 1 Loss of Pals1 in a colorectal cancer cell line HCT116 results in TJ defects, enhanced migration and invasion. A Immunostaining of confluent HCT116 and HCT116ΔPals1 cells with indicated antibodies, B Activation of Rac1 in wild type and Pals1-deficient HCT116 was quantified using G-LISA assay. C Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in HCT116 and HCT116ΔPals1 cells. Results are representative of 4 experiments. D Quantification of Rac1 biosensor FRET signals at the cell body and the cell cortex in HCT116 and HCT116ΔPals1 cells. Scale bars are 20 µm in A, C.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Activation Assay, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 2 Deletion of Pals1 in Caco-2 cells does not result in enhanced migration and invasion or upregulation of active Arf6 or Rac1. A Immunostaining of confluent Caco-2 and Caco-2ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of Caco-2 and Caco-2ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasions assays of Caco-2 and Caco-2ΔPals1 cells (N = 5). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from lysates of Caco-2 and Caco-2ΔPals1 cells (N = 3). F Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in Caco-2 and Caco-2ΔPals1 cells. Results are representative of 4 experiments. Scale bars are 20 µm in A and F, 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Migration, Immunostaining, Western Blot, Staining, Transfection

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 3 Pals1-deficient DLD1 do not exhibit increased Arf6/Rac1 activity or enhanced cell migration/invasion. A Immunostaining of confluent DLD1 and DLD1ΔPals1 cells with the indicated antibodies. B Representative images from wound healing assays of DLD1 and DLD1ΔPals1 cells and the corresponding quantification (N = 3). C Representative images and quantification of transwell matrigel invasion assays of DLD1 and DLD1ΔPals1 cells (N = 3). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 8). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of DLD1 and DLD1ΔPals1 cells (N = 3). Scale bars are 20 µm in A and 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activity Assay, Migration, Immunostaining, Western Blot, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 4 Knockout of Pals1 in mesenchymal-like RKO cells does not affect cell motility. A Western blot analysis of the expression of E-Cadherin in different colorectal cancer cell lines. B Representative images from wound healing assays of RKO and RKOΔPals1 cells and the corresponding quantification (N = 6). C Representative images and quantification of transwell matrigel invasion assays of RKO and RKOΔPals1 cells (N = 4). D Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of RKO and RKOΔPals1 cells (N = 6). E Western blot and CBB-stained gel of pulldown experiments to detect active Rac1 from cell lysates of RKO and RKOΔPals1 cells (N = 3). Scale bars are 100 µm in B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Knock-Out, Western Blot, Expressing, Staining

Journal: Cancer gene therapy

Article Title: Pals1 functions in redundancy with SMAP1 to inhibit Arf6 in order to prevent Rac1-dependent colorectal cancer cell migration and invasion.

doi: 10.1038/s41417-022-00570-2

Figure Lengend Snippet: Fig. 6 SW48ΔPals1 cells display enhanced Arf6/Rac1 activation and increased cell migration, which can be rescued by SMAP1 transfection. A Immunostaining of confluent SW48 and SW48ΔPals1 cells with the indicated antibodies. B Representative images and quantification of the FRET signal of a biosensor targeting active Rac1, transfected in SW48 and SW48ΔPals1 cells. Results are representative of 3 experiments. C Western blot of cell lines with and without SMAP1 overexpression. Empty vector was used as negative control. D, E Quantification of cell migration (scratch assay, D) and invasions assay (E) of the indicated cell lines. F Rac1 activation of the indicated cell lines quantified by G-LISA. G Western blot and CBB-stained gel of pulldown experiments to detect active Arf6 from cell lysates of the indicated cell lines (N = 3). H Survival probability of colorectal cancer patients with only low Pals1 expression, only low SMAP1 expression or low Pals1 and low SMAP1 expression. Scale bars are 20 µm in A and B.

Article Snippet: The Rac1 G-LISA activity assay (#BK128, Cytoskeleton Inc.) was performed according to manufacturer’s instructions, using 0.5 mg/ml protein concentration of cell lysates.

Techniques: Activation Assay, Migration, Transfection, Immunostaining, Western Blot, Over Expression, Plasmid Preparation, Negative Control, Wound Healing Assay, Staining, Expressing

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

doi: 10.1167/iovs.15-18974

Figure Lengend Snippet: RAC1 activity is increased in Tg-CA-RAC1 mice. RAC1 activity (GTP-bound active RAC1) was assessed in retinas by using a RAC1 activation assay (see Methods). Luminescence values of the RAC1-GTP in each sample at Pw2 were normalized to luminescence values of the RAC1-GTP in WT controls. Relative RAC1 activity in Tg-CA-RAC1 retinas is represented as a fold change compared to WT controls as shown in the bar graph (4.9 ± 1.8, * P < 0.05 , n = 3).

Article Snippet: GTP-bound active RAC1 levels were determined by using G-LISA RAC1 Activation Assay Kit (Cytoskeleton, Denver, CO, USA) containing an ELISA plate coated with a RAC-GTP binding protein.

Techniques: Activity Assay, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

doi: 10.1167/iovs.15-18974

Figure Lengend Snippet: Transgenic-CA-RAC1 mice develop progressive photoreceptor degeneration from Pw3 to Pw13. ( A ) Retinal sections stained by hematoxylin-eosin and ( B ) row counts of nuclei across the ONL width in WT ( a ) and Tg-CA-RAC1 mice at several postnatal times (Pw3 [ b ], Pw8 [ c ], and Pw13 [ d ]) showed progressive photoreceptor cell loss with aging (* P < 0.01, n = 4). Scale bar : 20 μm. ( C ) Retinal sections of WT ( e ) and Tg-CA-RAC1 mice at Pw3 ( f ), Pw8 ( g ), and Pw13 ( h ) by TUNEL assay. ( D ) TUNEL-positive ( red ) cell counts indicated increased apoptosis signals in the ONL at all three ages as compared with WT controls, which showed nearly no TUNEL-positive cells in any layers. Scale bar : 20 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: GTP-bound active RAC1 levels were determined by using G-LISA RAC1 Activation Assay Kit (Cytoskeleton, Denver, CO, USA) containing an ELISA plate coated with a RAC-GTP binding protein.

Techniques: Transgenic Assay, Staining, TUNEL Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

doi: 10.1167/iovs.15-18974

Figure Lengend Snippet: Apocynin, a NOX inhibitor, reduces superoxide accumulation in Tg-CA-RAC1 retinas. Transgenic-CA-RAC1 mice were given daily IP injections of apocynin or vehicle control for ∼3 weeks starting at P4. At the end of Pw3, mice were injected intraperitoneally with DHE (20 mg/kg) and euthanized after 18 hours. Retinal sections were examined by confocal microscopy. ( A ) Representative images of the retinal sections are shown. Compared to the WT control ( a , b ), uninjected Tg-CA-RAC1 retina ( c , d ) had stronger oxidized DHE staining ( red ) in the ONL layer. Apocynin-injected Tg-CA-RAC1 retina ( g , h ) showed much less red staining than vehicle-injected Tg-CA-RAC1 retina ( e , f ), which had similar red staining to that shown in uninjected Tg-CA-RAC1 retina ( c , d ). Scale bar : 50 μm. ( B ) Dihydroethidium fluorescence intensity of the ONL region was measured in the WT, uninjected Tg-CA-RAC1, vehicle-injected Tg-CA-RAC1, and apocynin-injected Tg-CA-RAC1 retinas. Dihydroethidium intensity values of each sample were normalized to DHE values of WT samples. Quantification of DHE intensity is represented as a fold change compared to WT retinas in the bar graph (** P < 0.001, n = 3). The data indicate that NOX contributes to superoxide production in Tg-CA-RAC1 retinas.

Article Snippet: GTP-bound active RAC1 levels were determined by using G-LISA RAC1 Activation Assay Kit (Cytoskeleton, Denver, CO, USA) containing an ELISA plate coated with a RAC-GTP binding protein.

Techniques: Transgenic Assay, Injection, Confocal Microscopy, Staining, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

doi: 10.1167/iovs.15-18974

Figure Lengend Snippet: Apocynin attenuates oxidative damage in Tg-CA-RAC1 retinas. Transgenic-CA-RAC1 mice were given daily IP injections of apocynin or vehicle control for ∼7 weeks starting at P4. Mice were euthanized at Pw7. ( A ) Equal amounts of proteins from retinal extracts of vehicle-injected (Apo − ) and apocynin-injected (Apo + ) Tg-CA-RAC1 mice were analyzed by Western blot for protein carbonylation, a marker for oxidative stress. Western blots with anti-DNP antibody show carbonylated proteins in retinal extracts of Tg-CA-RAC1 (Apo − and Apo + ) mice. Actin is used as an additional control for Western blot. ( B ) Fluorescence values of each carbonylated protein band (180, 150, 100, 75, 60, 50, 37, 25 kDa in [ A ]) were normalized to fluorescence values of corresponding actin band and then totaled to provide an overall estimate. Total protein carbonyl content of apocynin-injected Tg-CA-RAC1 (Apo + ) retinas is represented as a fold change compared to that in vehicle-injected Tg-CA-RAC1 (Apo − ) retinas (0.5 ± 0.08, * P < 0.01, n = 3). ( C ) A TBARS assay was performed to measure the levels of MDA, a marker of oxidative stress and lipid peroxidation, in vehicle-injected (Apo − ) and apocynin-injected (Apo + ) Tg-CA-RAC1 retinas. Data are represented as a fold change in MDA concentrations compared to those in vehicle-injected Tg-CA-RAC1 (Apo − ) retinas (0.39 ± 0.04, * P < 0.01, n = 3). Both results indicate that NOX activation contributes to oxidative damage in Tg-CA-RAC1 retinas.

Article Snippet: GTP-bound active RAC1 levels were determined by using G-LISA RAC1 Activation Assay Kit (Cytoskeleton, Denver, CO, USA) containing an ELISA plate coated with a RAC-GTP binding protein.

Techniques: Transgenic Assay, Injection, Western Blot, Marker, Fluorescence, TBARS Assay, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

doi: 10.1167/iovs.15-18974

Figure Lengend Snippet: Expression of CA-RAC1 in adult WT photoreceptors induces photoreceptor degeneration. AAV8- pOpsin-CA Rac1-GFP or control AAV8- pOpsin-GFP vectors were delivered subretinally to Pw6 WT eyes. Retinal morphology was evaluated 10 weeks after injections. Retinal sections were immunostained with antibody against MYC-tag ( red , CA RAC1). Retinal nuclei were labeled with DAPI ( blue ). In total, four animals for each vector were examined. As shown in representative cross-sections of AAV8 -pOpsin-GFP –injected ( A ) and AAV8- pOpsin-CA Rac1-GFP –injected ( B ) retinas, at least one-third of the entire length of the ONL regions was transduced by AAV8 vectors. In AAV8- pOpsin-CA Rac1-GFP –injected animals ( B ), both CA RAC1-GFP –transduced ( b , c , d ; yellow ) and non– CA RAC1-GFP -transduced ( e , f ) ONL regions were observed in a single retina. Thus, the ONL regions with no CA Rac1 - GFP –transduced photoreceptors serve as internal controls. Scale bar : 200 μm. ( C ) High magnification images of regions ([ a in A ] and [ b , c , d , e , f in B ]) are shown. Scale bar : 50 μm. In AAV8- pOpsin-CA Rac1-GFP –injected retinas ( B ), CA Rac1-GFP –transduced regions ( b , c , d ; yellow ) show significant ONL cell loss (compare [ b , c , d in B ] with [ a in A ]). In contrast, no cell loss was observed in non– CA Rac1-GFP -transduced regions ( e , f ), which had similar ONL thickness to those in control GFP -transduced regions ( a ). ( D ) The ONL thickness was evaluated as described in Methods. Average row counts of nuclei across the ONL width are shown in control GFP -transduced (AAV8- pOpsin-GFP ), CA Rac1-GFP –transduced, and non– CA Rac1-GFP -transduced regions (** P < 0.001, n = 4). There are significantly fewer ONL nuclei in CA Rac1 –transduced regions than in control GFP -transduced regions. In AAV8- pOpsin-CA Rac1-GFP –injected retinas, CA Rac1 –transduced regions show significantly fewer ONL nuclei than non– CA Rac1 -transduced regions.

Article Snippet: GTP-bound active RAC1 levels were determined by using G-LISA RAC1 Activation Assay Kit (Cytoskeleton, Denver, CO, USA) containing an ELISA plate coated with a RAC-GTP binding protein.

Techniques: Expressing, Labeling, Plasmid Preparation, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

doi: 10.1167/iovs.15-18974

Figure Lengend Snippet: Apocynin rescues photoreceptor cells and rod function. Transgenic-CA-RAC1 mice were intraperitoneally injected daily with apocynin or vehicle control starting at P4 and euthanized at Pw13. ( A ) Retinal morphology is shown by toluidine blue–stained plastic sections cut through the ONH along the vertical meridian. Scale bar : 20 μm. ( B ) The ONL thickness is evaluated by counting rows of nuclei across the ONL width at 10 points along the retinal length of plastic sections containing ONH. Apocynin-injected Tg-CA-RAC1 mice had significantly more photoreceptor cells than vehicle-injected Tg-CA-RAC1 mice ( P < 0.01, n = 4). ( C ) Retinal responses were measured by dark-adapted ERG before the mice were euthanized. Amplitude versus log intensity (V-log I) curve of dark-adapted ERG recordings shows that apocynin-injected Tg-CA-RAC1 mice had significantly larger a- and b-wave amplitudes than vehicle-injected mice ( P < 0.01 and P < 0.01, respectively; n = 4).

Article Snippet: GTP-bound active RAC1 levels were determined by using G-LISA RAC1 Activation Assay Kit (Cytoskeleton, Denver, CO, USA) containing an ELISA plate coated with a RAC-GTP binding protein.

Techniques: Transgenic Assay, Injection, Staining

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a KRAS Mut cell lines (H358, A427, NCIH727, NCIH23 and SKLU-1), EGFR Mut cell lines (HCC827, HCC2279, H1650 and H1975), KRAS WT and EGFR WT cell lines (H322M, H522, Calu-3 and HCC1666) and nontumorigenic cells (HEK-293T and BEAS-2B) were collected with lysis buffer, and SIRT1 activity was measured with cell lysates. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. b Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines used in a were collected with lysis buffer, and immunoblotted with anti-pMKK4, MKK, pMKK7, MKK, pJNK1, JNK1 and β-actin antibodies. c KRAS Mut cell lines (H358, A427 and NCIH727) were treated with anisomycin 38 μM (10 μg/ml) (JNK1 activator) and SP600125 20 μM (JNK1 inhibitor) for 2 h. The protein levels of pJNK1, JNK, pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 , SIRT1 and β-actin were measured by western blot analysis. d H358 cells were treated with anisomycin and SP600125 under the same condition as in b and then whole-cell lysates were subjected to immunoprecipitation with an anti-JNK1 antibody. Immunoblot analysis was performed using antibodies against SIRT1, pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 , pJNK1, JNK and β-actin antibodies. e The recombinant proteins, SIRT1 and JNK1 were incubated in the reaction mixture for phosphorylation at 32 °C for 4 h, and then Ser- and Thr-phosphorylated peptides were identified using anti-pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 antibody. f H358 cells were transfected with SIRT1 WT , SIRT1 T530A , SIRT1 S27A&S47A , and then H358 cell extracts were immunoprecipitated with anti-KRAS antibody and RAF-1 agarose beads and immunoblotted with anti-acetylation, anti-SIRT1, anti-KRAS, anti-KRAS–GTP-bound and β-actin antibodies. g KRAS Mut cell lines (H358, A427 and H727) were transfected with SIRT1 WT , SIRT1 S27A , SIRT1 S47A , SIRT1 T530A and SIRT1 S27A,S47A (2 μg), then collected with lysis buffer, and SIRT1 activity was assessed with cell lysates. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: Lysis, Activity Assay, Western Blot, Immunoprecipitation, Recombinant, Incubation, Phospho-proteomics, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines (H358, A427 and H727) were collected with lysis buffer and subjected to western blotting with anti-pERK, ERK and β-actin antibodies. b A luciferase assay was performed to assess the AP-1-mediated transcriptional regulatory activity with cell lysates in Fig. 4a. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. c TGFB1 mRNA expression was measured by RT–qPCR with same cell lines as in a . RPL32 was used as internal control and for normalization. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. d The medium of four cell lines were changed by FBS-free medium before cell collection at 24 h. Conditioned medium was collected and concentrated using an Amicon Ultra-15 tube, and total TGF-β1 levels were measured by ELISA. e KRAS Mut cell lines (H358, A427 and H727) were transfected with pcDNA , KRAS G12C , G12D and G12V plasmids (2 μg). TGF-β1 levels were measured under the same method as in d . f H358, A427 and H727 cells were transplanted with pcDNA , KRAS G12C , G12D and G12V plasmids, siCon and siSmad2/3 (80 nM) for 48 h, and then the activity of Smad2/3, JNK1 and KRAS was measured. g The cell lysates of each different KRAS Mut cell lines (H358, A427 and H727) under KWN-C with indicated dosage for 24 h were transferred by immunoblotting assay with anti-pSmad2/3, anti-Smad2/3, pJNK1, JNK1, anti-pSIRT1 Ser27 , pSIRT1 Ser47 , SIRT1, KRAS–GTP-bound and β-actin antibodies. h , H358, A427 and H460 cells were treated with DMSO or KWN-C (10 μM), and cell extracts were then immunoprecipitated using immunoglobulin G, anti-KRAS, and RAF-1 agarose bead antibodies. Immunoblotting was performed using anti-acetyl, anti-KRAS–GTP-bound, anti-SIRT1, anti-KRAS and β-actin antibodies.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: Lysis, Western Blot, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a H358, H460, NCIH23, SKLU-1 and SW900 cells were treated with CP (H358 1 μM, H460, NCIH23 and SKLU-1 5 μM), MTA (H358 10 μM, H460, NCIH23 and SKLU-1 5 μM) and/or KWN-C (10 μM), and cell proliferation was measured by the MTS assay 3 days after drug treatment. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. b Top: KRAS Mut cells were seeded with 0.5% top agar and cultured in a mixture of fresh medium with drugs as described in Supplementary Fig. . Cell colonies were stained with crystal violet and counted per 3.8 cm 2 . Bottom: representative colony images are shown. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. c H358 and H460 cells were treated with CP (H358 1 μM and H460 5 μM), MTA (H358 10 μM and H460 5 μM) and/or KWN-C (10 μM). Cell lysates from drug-treated cells were incubated with Raf-1–RBD to pull down KRAS–GTP (the active form of KRAS), followed by western blotting with an anti-KRAS antibody. Expression levels of KRAS, pERK, ERK, pAkt, Akt, PARP, cleaved PARP, pro-caspase-3, cleaved-caspase-3 and β-actin in total lysates were analyzed by western blotting. d H358 and H460 cells treated as in a were assessed for apoptosis by TUNEL assay (middle row), and their nuclei were stained with DAPI (top row; scale bar, 25 μm). All figures are representative of at least three separate experiments. e H358 and H460 cells treated as in a were stained with Annexin V/PI staining for apoptosis using flow cytometric analysis. Representative flow cytometry plots. All figures are representative of at least three separate experiments.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: MTS Assay, Cell Culture, Staining, Incubation, Western Blot, Expressing, TUNEL Assay, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a H358 cells harboring stably expressed luciferase plasmid were intratracheally injected into nude mice (1 × 10 6 cells per mouse). Top: representative bioluminescence images 2 months after the injection. The mice were euthanized 2 months after the injection, and lungs were excised and stained with Bouin’s fixative. Bottom: the lung tumor images. Therapeutic candidates were treated with CP (5 mg/kg per day, i.p.), MTA (150 mg/kg twice a week, i.p.) and/or KWN-C (30 mg/kg per day, i.p.). b The photon emission values represent the mean ± s.e.m. of the indicated number of mice. Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. c The lung tumor weight from combination CP, MTA and/or KWN-C-treated mice was measured and compared with nontreatment, each single treatment and combined treatment. Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. d The number of colonies formed in the lungs were measured under microscopy under the same conditions as in c . Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. e KRAS–GTP (active form of KRAS) was pulled down by Raf-1–RBD from tumor tissue lysates, followed by western blot using KRAS antibody. Expression levels of anti-pSIRT1 S27 , pSIRT1 S47 , SIRT1, KRAS–GTP-bound, KRAS, pERK, ERK pAkt, Akt, PARP, cleaved PARP, pro-caspase-3, cleaved-caspase-3 and β-actin were analyzed by western blot in tumor tissues.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Luciferase, Plasmid Preparation, Injection, Staining, Microscopy, Western Blot, Expressing