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: Oncogene

Article Title: Upregulation of PIP3-Dependent Rac Exchanger 1 (P-Rex1) Promotes Prostate Cancer Metastasis

doi: 10.1038/onc.2009.30

Figure Lengend Snippet: P-Rex1 promotes prostate cancer cell migration and invasion via Rac activation. (a) CWR22Rv1 cells (left panel) or PC3-LN4 cells (right panel) were transfected with control GFP, GFP-tagged wild-type P-Rex1 or P-Rex1(-DH) mutant for 48 h, and then subjected to Transwell migration assay for 12 h (CWR22Rv1 cells) or 5 h (PC3-LN4) toward NIH3T3 CM. Transfected PC3-LN4 cells were selected by flow cytometry cell sorting based on GFP fluorescence prior to migration assays due to a low transfection efficiency (~25%). Bars show mean ± S.E. with *p<0.05 compared to control set at 100% (n=3). Inset: Expression of GFP-tagged P-Rex1 and P-Rex1(-DH) protein detected by western blot analysis using anti-GFP antibody. (b) CWR22Rv1 cells stably expressing wild-type P-Rex1, P-Rex1 “GEF-dead” mutant or control vector were subjected to migration and invasion assays for 12 h. Cells stably expressing wild-type P-Rex1 were pretreated without (none) or with 50 μM NSC for 15 min. Bars show mean ± S.E. with *p<0.01 compared to control (n=3). Inset: Western blot analysis of expression levels of P-Rex1 and its mutant. (c) P-Rex1 activates Rac1 in prostate cancer cells. The amount of activated GTP-bound Rac1 was measured by a Rac activation assay. The image shown is representative of 3 separate experiments. (d) P-Rex1 increases lamellipodia formation. Serum-starved CWR22Rv1 cells or PC-3 cells (positive control) were incubated with NIH3T3 CM for 5 h and then stained with rhodamine-conjugated phalloidin (red) for detection of F-actin.

Article Snippet: Additional reagents were NSC23766, Y27632 and anti-GFP antibody (EMD Biosciences); cell permeable C3 transferase and F-actin visualization kit (Cytoskeleton); Rac activation assay kit, Rho assay reagent, anti-Rac1 antibody (clone 23A8), and Immobilon-FL (Millipore); mouse anti-RhoA (26C4), and rabbit anti-HA and anti-β-actin antibodies (Santa Cruz); FBS (Hyclone); Lipofectamine TM 2000, AlexaF488-labeled secondary antibody, RPMI-1640 and DMEM (Invitrogen); EGF, Matrigel and fibronectin (BD Biosciences); CXCL12 (R&D Systems); and IRDye-labeled secondary antibodies (LI-COR Biosciences).

Techniques: Migration, Activation Assay, Transfection, Control, Mutagenesis, Transwell Migration Assay, Flow Cytometry, FACS, Fluorescence, Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Positive Control, Incubation, Staining

Journal: Journal of Biological Chemistry

Article Title: RACK1 Regulates VEGF/Flt1-mediated Cell Migration via Activation of a PI3K/Akt Pathway

doi: 10.1074/jbc.m110.165605

Figure Lengend Snippet: FIGURE 7. RACK1-silencing significantly decreased VEGF-induced small-GTPase Rac1 activation and membrane ruffling. A, AG1-G1-Neo and AG1-G1- Flt1 cells transfected with control siRNA (si-ctl) or RACK1 siRNAs (si1 and si2) were serum-starved and stimulated with VEGF (100 ng/ml) for 3 min. Cell lysate was subjected to a pull-down assay with GST-PAK-PBD (GST-fused GTPase-binding domain) agarose beads, followed by Western blotting with anti-Rac1 antibody. RACK1-silencing remarkably decreased VEGF-induced activation of Rac1. Cell lysates were immunoblotted with antibodies against Rac1, and -actin as a control. B, AG1-G1-Neo and AG1-G1-Flt1 cells transfected with control siRNA (si-ctl) or RACK1 siRNAs (si1 and si2) were serum-starved and stimulated with VEGF (100ng/ml)for5min.Cellswerefixed,permeabilized,andstainedwithrhodamine-phalloidin(F-actin).Cellmorphologywasobservedbyconfocalmicroscope, and representative images are presented (upper panels). Scale bar, 10 m. The numbers of cells exhibiting membrane ruffling were counted (lower panels). RACK1-silencing significantly inhibited VEGF-induced membrane ruffling.

Article Snippet: Rac1 Activation Assay—The activation of Rac1 was analyzed by using a Rac1 activation assay kit (Cytoskeleton, Denver, CO) according to the instructions recommended by the manufacturer.

Techniques: Activation Assay, Membrane, Transfection, Control, Pull Down Assay, Binding Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: RACK1 Regulates VEGF/Flt1-mediated Cell Migration via Activation of a PI3K/Akt Pathway

doi: 10.1074/jbc.m110.165605

Figure Lengend Snippet: FIGURE 8. The possible role of RACK1 in VEGF/Flt1-induced cell migra- tion. By interacting with Flt1, RACK1 plays an important role in VEGF/Flt1- induced cell migration by regulating the activation of the PI3K/Akt but not PLC-MAPK signaling pathway. RACK1 regulates the VEGF/Flt1-induced acti- vation of Rac1 and cytoskeletal dynamics.

Article Snippet: Rac1 Activation Assay—The activation of Rac1 was analyzed by using a Rac1 activation assay kit (Cytoskeleton, Denver, CO) according to the instructions recommended by the manufacturer.

Techniques: Migration, Activation Assay

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A) Effects of CD148 in E-cadherin contact formation were examined by a calcium-switch assay and immunofluorescence staining. CD148 WT, but not CS, promotes E-cadherin contact formation in A431D/E-cadherin WT cells, as evidenced by the expanded and intense E-cadherin distribution (left panels), while CD148 WT shows no effects in A431D/E-cadherin 764 AAA cells (right panels). B) Activities of Rac1, Cdc42, and RhoA were measured in the condition of calcium switch assay. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by the pull-down assays and/or immunoblotting described in the “ ” (left panels). The activity was normalized to total amount of protein using densitometry and expressed as fold change (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A and B) CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells. CD148 WT, but not CS, increases Rac1 activity in A431D/E-cadherin WT cells, while this effect is not observed in A431D/E-cadherin 764 AAA cells. C) Effects of CD148 WT in cell-cell adhesion were assessed by a hanging drop assay in the presence or absence of Rac1 inhibitor NSC 23766 (75 µM). CD148 increase of cell-cell adhesion is largely diminished by Rac1 inhibition.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Western Blot, Activity Assay, Inhibition

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