111224_PCR.xlsx

Selected Cell

Cell:

Value:

Raw XBP1 data Raw TRIB3 and HSPA5 data Processing Analyzed data Methods

Replicate and treatment are in the sample name column. 1 = replicate 1, 2 = replicate 2, 3 = replicate 3, m = replicate 4
NT = not treated, tun = tunicamycin treated, 4u = 4u8c treated
Replicates 1 and 2
Well	Well Position	Omit	Sample Name	Target Name	Task	Reporter	Quencher	RQ	RQ Min	RQ Max	CT	Ct Mean	Ct SD	Quantity	Delta Ct Mean	Delta Ct SD	Delta Delta Ct	Automatic Ct Threshold	Ct Threshold	Automatic Baseline	Baseline Start	Baseline End	Efficiency	Comments	Tm1	Tm2	Tm3	Custom1	Custom2	Custom3	Custom4	Custom5	Custom6	MTP
1	A1	false	1nt	xbp1 spliced	UNKNOWN	SYBR	None	1	0.8182626962661743	1.2221014499664307	30.288999557495117	30.415618896484375	0.10971656441688538		8.589860916137695	0.18051615357398987	0	true	0.10575682687542884	true	3	27	1		84.55587005615234									N
2	A2	false	1nt	xbp1 spliced	UNKNOWN	SYBR	None	1	0.8182626962661743	1.2221014499664307	30.476999282836914	30.415618896484375	0.10971656441688538		8.589860916137695	0.18051615357398987	0	true	0.10575682687542884	true	3	27	1		84.55587005615234									N
3	A3	false	1nt	xbp1 spliced	UNKNOWN	SYBR	None	1	0.8182626962661743	1.2221014499664307	30.481000900268555	30.415618896484375	0.10971656441688538		8.589860916137695	0.18051615357398987	0	true	0.10575682687542884	true	3	27	1		84.55587005615234									N
7	A7	false	2nt	xbp1 spliced	UNKNOWN	SYBR	None	0.9528027772903442	0.8740479946136475	1.0386537313461304	30.617000579833984	30.56184959411621	0.06257332861423492		8.659611701965332	0.07764605432748795	0.06975046545267105	true	0.10575682687542884	true	3	27	1		84.75274658203125									N
8	A8	false	2nt	xbp1 spliced	UNKNOWN	SYBR	None	0.9528027772903442	0.8740479946136475	1.0386537313461304	30.493999481201172	30.56184959411621	0.06257332861423492		8.659611701965332	0.07764605432748795	0.06975046545267105	true	0.10575682687542884	true	3	27	1		84.65430450439453									N
9	A9	false	2nt	xbp1 spliced	UNKNOWN	SYBR	None	0.9528027772903442	0.8740479946136475	1.0386537313461304	30.575000762939453	30.56184959411621	0.06257332861423492		8.659611701965332	0.07764605432748795	0.06975046545267105	true	0.10575682687542884	true	3	27	1		84.55587005615234									N
49	E1	false	1tun	xbp1 spliced	UNKNOWN	SYBR	None	145.83819580078125	141.48167419433594	150.328857421875	24.365999221801758	24.386056900024414	0.017452871426939964		1.4016361236572266	0.02729501947760582	-7.188224792480469	true	0.10575682687542884	true	3	20	1		84.55587005615234									N
50	E2	false	1tun	xbp1 spliced	UNKNOWN	SYBR	None	145.83819580078125	141.48167419433594	150.328857421875	24.39299964904785	24.386056900024414	0.017452871426939964		1.4016361236572266	0.02729501947760582	-7.188224792480469	true	0.10575682687542884	true	3	20	1		84.65430450439453									N
51	E3	false	1tun	xbp1 spliced	UNKNOWN	SYBR	None	145.83819580078125	141.48167419433594	150.328857421875	24.39900016784668	24.386056900024414	0.017452871426939964		1.4016361236572266	0.02729501947760582	-7.188224792480469	true	0.10575682687542884	true	3	20	1		84.55587005615234									N
55	E7	false	2tun	xbp1 spliced	UNKNOWN	SYBR	None	204.15867614746094	176.13702392578125	236.6383056640625	22.954999923706055	22.971158981323242	0.037734150886535645		0.9163138270378113	0.13287286460399628	-7.673547267913818	true	0.10575682687542884	true	3	19	1		84.55587005615234									N
56	E8	false	2tun	xbp1 spliced	UNKNOWN	SYBR	None	204.15867614746094	176.13702392578125	236.6383056640625	22.944000244140625	22.971158981323242	0.037734150886535645		0.9163138270378113	0.13287286460399628	-7.673547267913818	true	0.10575682687542884	true	3	18	1		84.45743560791016									N
57	E9	false	2tun	xbp1 spliced	UNKNOWN	SYBR	None	204.15867614746094	176.13702392578125	236.6383056640625	23.013999938964844	22.971158981323242	0.037734150886535645		0.9163138270378113	0.13287286460399628	-7.673547267913818	true	0.10575682687542884	true	3	19	1		84.45743560791016									N
85	H1	false	1tun4u	xbp1 spliced	UNKNOWN	SYBR	None	0.052415881305933	0.03492182493209839	0.07867357134819031	32.9630012512207	33.34567642211914	0.36268433928489685		12.84371280670166	0.3654909133911133	4.253852367401123	true	0.10575682687542884	true	3	28	1		84.35900115966797									N
86	H2	false	1tun4u	xbp1 spliced	UNKNOWN	SYBR	None	0.052415881305933	0.03492182493209839	0.07867357134819031	33.388999938964844	33.34567642211914	0.36268433928489685		12.84371280670166	0.3654909133911133	4.253852367401123	true	0.10575682687542884	true	3	28	1		84.45743560791016									N
87	H3	false	1tun4u	xbp1 spliced	UNKNOWN	SYBR	None	0.052415881305933	0.03492182493209839	0.07867357134819031	33.685001373291016	33.34567642211914	0.36268433928489685		12.84371280670166	0.3654909133911133	4.253852367401123	true	0.10575682687542884	true	3	28	1		84.45743560791016									N
91	H7	false	2tun4u	xbp1 spliced	UNKNOWN	SYBR	None	0.035957515239715576	0.021047404035925865	0.06143003702163696	35.50699996948242	35.02152633666992	0.4690195322036743		13.387423515319824	0.4820087254047394	4.797563076019287	true	0.10575682687542884	true	3	29	1		84.65430450439453									N
92	H8	false	2tun4u	xbp1 spliced	UNKNOWN	SYBR	None	0.035957515239715576	0.021047404035925865	0.06143003702163696	34.98699951171875	35.02152633666992	0.4690195322036743		13.387423515319824	0.4820087254047394	4.797563076019287	true	0.10575682687542884	true	3	29	1		84.55587005615234									N
93	H9	false	2tun4u	xbp1 spliced	UNKNOWN	SYBR	None	0.035957515239715576	0.021047404035925865	0.06143003702163696	34.57099914550781	35.02152633666992	0.4690195322036743		13.387423515319824	0.4820087254047394	4.797563076019287	true	0.10575682687542884	true	3	29	1		84.55587005615234									N
4	A4	false	1nt	xbp1 unspliced	UNKNOWN	SYBR	None				21.929000854492188	21.825759887695312	0.14334698021411896					true	0.1751817209411774	true	3	17	1		83.96526336669922									N
5	A5	false	1nt	xbp1 unspliced	UNKNOWN	SYBR	None				21.88599967956543	21.825759887695312	0.14334698021411896					true	0.1751817209411774	true	3	17	1		83.96526336669922									N
6	A6	false	1nt	xbp1 unspliced	UNKNOWN	SYBR	None				21.66200065612793	21.825759887695312	0.14334698021411896					true	0.1751817209411774	true	3	16	1		84.0636978149414									N
10	A10	false	2nt	xbp1 unspliced	UNKNOWN	SYBR	None				21.937999725341797	21.902236938476562	0.04597269371151924					true	0.1751817209411774	true	3	16	1		83.86682891845703									N
11	A11	false	2nt	xbp1 unspliced	UNKNOWN	SYBR	None				21.91900062561035	21.902236938476562	0.04597269371151924					true	0.1751817209411774	true	3	16	1		83.86682891845703									N
12	A12	false	2nt	xbp1 unspliced	UNKNOWN	SYBR	None				21.850000381469727	21.902236938476562	0.04597269371151924					true	0.1751817209411774	true	3	16	1		83.86682891845703									N
52	E4	false	1tun	xbp1 unspliced	UNKNOWN	SYBR	None				22.9950008392334	22.984420776367188	0.020986076444387436					true	0.1751817209411774	true	3	18	1		83.96526336669922									N
53	E5	false	1tun	xbp1 unspliced	UNKNOWN	SYBR	None				22.99799919128418	22.984420776367188	0.020986076444387436					true	0.1751817209411774	true	3	18	1		83.96526336669922									N
54	E6	false	1tun	xbp1 unspliced	UNKNOWN	SYBR	None				22.959999084472656	22.984420776367188	0.020986076444387436					true	0.1751817209411774	true	3	18	1		83.96526336669922									N
58	E10	false	2tun	xbp1 unspliced	UNKNOWN	SYBR	None				22.104000091552734	22.054845809936523	0.12740224599838257					true	0.1751817209411774	true	3	17	1		83.86682891845703									N
59	E11	false	2tun	xbp1 unspliced	UNKNOWN	SYBR	None				22.149999618530273	22.054845809936523	0.12740224599838257					true	0.1751817209411774	true	3	17	1		83.86682891845703									N
60	E12	false	2tun	xbp1 unspliced	UNKNOWN	SYBR	None				21.90999984741211	22.054845809936523	0.12740224599838257					true	0.1751817209411774	true	3	16	1		83.96526336669922									N
88	H4	false	1tun4u	xbp1 unspliced	UNKNOWN	SYBR	None				20.545000076293945	20.50196647644043	0.045207127928733826					true	0.1751817209411774	true	3	15	1		83.86682891845703									N
89	H5	false	1tun4u	xbp1 unspliced	UNKNOWN	SYBR	None				20.5049991607666	20.50196647644043	0.045207127928733826					true	0.1751817209411774	true	3	15	1		83.86682891845703									N
90	H6	false	1tun4u	xbp1 unspliced	UNKNOWN	SYBR	None				20.454999923706055	20.50196647644043	0.045207127928733826					true	0.1751817209411774	true	3	15	1		83.96526336669922									N
94	H10	false	2tun4u	xbp1 unspliced	UNKNOWN	SYBR	None				21.594999313354492	21.634103775024414	0.11114447563886642					true	0.1751817209411774	true	3	16	1		83.76839447021484									N
95	H11	false	2tun4u	xbp1 unspliced	UNKNOWN	SYBR	None				21.54800033569336	21.634103775024414	0.11114447563886642					true	0.1751817209411774	true	3	16	1		83.66996002197266									N
96	H12	false	2tun4u	xbp1 unspliced	UNKNOWN	SYBR	None				21.759000778198242	21.634103775024414	0.11114447563886642					true	0.1751817209411774	true	3	17	1		83.66996002197266									N

Replicates 3 and 4
Well	Well Position	Omit	Sample Name	Target Name	Task	Reporter	Quencher	RQ	RQ Min	RQ Max	CT	Ct Mean	Ct SD	Quantity	Delta Ct Mean	Delta Ct SD	Delta Delta Ct	Automatic Ct Threshold	Ct Threshold	Automatic Baseline	Baseline Start	Baseline End	Efficiency	Comments	Tm1	Tm2	Tm3	Custom1	Custom2	Custom3	Custom4	Custom5	Custom6	HIGHSD	MTP
1	A1	false	3nt	xbp1 sp	UNKNOWN	SYBR	None	1	0.930397629737854	1.0748093128204346	32.29600143432617	32.285579681396484	0.061162084341049194		8.322842597961426	0.06492944806814194	0	true	0.11800127860501854	true	3	27	1		84.56365966796875									N	N
2	A2	false	3nt	xbp1 sp	UNKNOWN	SYBR	None	1	0.930397629737854	1.0748093128204346	32.340999603271484	32.285579681396484	0.061162084341049194		8.322842597961426	0.06492944806814194	0	true	0.11800127860501854	true	3	27	1		84.56365966796875									N	N
3	A3	false	3nt	xbp1 sp	UNKNOWN	SYBR	None	1	0.930397629737854	1.0748093128204346	32.220001220703125	32.285579681396484	0.061162084341049194		8.322842597961426	0.06492944806814194	0	true	0.11800127860501854	true	3	27	1		84.56365966796875									N	N
7	A7	false	mnt	xbp1 sp	UNKNOWN	SYBR	None	1.2887864112854004	1.072513461112976	1.548671007156372	30.6560001373291	30.83660888671875	0.15729603171348572		7.95682954788208	0.16532787680625916	-0.36601319909095764	true	0.11800127860501854	true	3	26	1		84.66212463378906									N	N
8	A8	false	mnt	xbp1 sp	UNKNOWN	SYBR	None	1.2887864112854004	1.072513461112976	1.548671007156372	30.94099998474121	30.83660888671875	0.15729603171348572		7.95682954788208	0.16532787680625916	-0.36601319909095764	true	0.11800127860501854	true	3	25	1		84.56365966796875									N	N
9	A9	false	mnt	xbp1 sp	UNKNOWN	SYBR	None	1.2887864112854004	1.072513461112976	1.548671007156372	30.913000106811523	30.83660888671875	0.15729603171348572		7.95682954788208	0.16532787680625916	-0.36601319909095764	true	0.11800127860501854	true	3	26	1		84.56365966796875									N	N
49	E1	false	3tun	xbp1 sp	UNKNOWN	SYBR	None	236.30685424804688	219.14183044433594	254.81637573242188	23.638999938964844	23.601655960083008	0.040742628276348114		0.43832525610923767	0.06787138432264328	-7.884517669677734	true	0.11800127860501854	true	3	19	1		84.66212463378906									N	N
50	E2	false	3tun	xbp1 sp	UNKNOWN	SYBR	None	236.30685424804688	219.14183044433594	254.81637573242188	23.558000564575195	23.601655960083008	0.040742628276348114		0.43832525610923767	0.06787138432264328	-7.884517669677734	true	0.11800127860501854	true	3	19	1		84.76058959960938									N	N
51	E3	false	3tun	xbp1 sp	UNKNOWN	SYBR	None	236.30685424804688	219.14183044433594	254.81637573242188	23.60700035095215	23.601655960083008	0.040742628276348114		0.43832525610923767	0.06787138432264328	-7.884517669677734	true	0.11800127860501854	true	3	19	1		84.66212463378906									N	N
55	E7	false	mtun	xbp1 sp	UNKNOWN	SYBR	None	231.7190704345703	214.57675170898438	250.23086547851562	23.3799991607666	23.342636108398438	0.04997463524341583		0.4666099548339844	0.06917297840118408	-7.8562331199646	true	0.11800127860501854	true	3	18	1		84.56365966796875									N	N
56	E8	false	mtun	xbp1 sp	UNKNOWN	SYBR	None	231.7190704345703	214.57675170898438	250.23086547851562	23.363000869750977	23.342636108398438	0.04997463524341583		0.4666099548339844	0.06917297840118408	-7.8562331199646	true	0.11800127860501854	true	3	19	1		84.56365966796875									N	N
57	E9	false	mtun	xbp1 sp	UNKNOWN	SYBR	None	231.7190704345703	214.57675170898438	250.23086547851562	23.285999298095703	23.342636108398438	0.04997463524341583		0.4666099548339844	0.06917297840118408	-7.8562331199646	true	0.11800127860501854	true	3	18	1		84.46520233154297									N	N
85	H1	false	3tun4uc	xbp1 sp	UNKNOWN	SYBR	None	0.03784313052892685	0.029094120487570763	0.04922308772802353	34.979000091552734	34.864803314208984	0.23662219941616058		13.04666805267334	0.23662395775318146	4.723824977874756	true	0.11800127860501854	true	3	30	1		84.36673736572266									N	N
86	H2	false	3tun4uc	xbp1 sp	UNKNOWN	SYBR	None	0.03784313052892685	0.029094120487570763	0.04922308772802353	34.59299850463867	34.864803314208984	0.23662219941616058		13.04666805267334	0.23662395775318146	4.723824977874756	true	0.11800127860501854	true	3	29	1		84.36673736572266									N	N
87	H3	false	3tun4uc	xbp1 sp	UNKNOWN	SYBR	None	0.03784313052892685	0.029094120487570763	0.04922308772802353	35.02299880981445	34.864803314208984	0.23662219941616058		13.04666805267334	0.23662395775318146	4.723824977874756	true	0.11800127860501854	true	3	30	1		84.46520233154297									N	N
91	H7	false	mtun4u	xbp1 sp	UNKNOWN	SYBR	None	0.040832120925188065	0.0209805928170681	0.07946687191724777	36.4739990234375	37.14285659790039	0.5987184643745422		12.936994552612305	0.5992890000343323	4.614151477813721	true	0.11800127860501854	true	3	31	1		84.66212463378906									Y	N
92	H8	false	mtun4u	xbp1 sp	UNKNOWN	SYBR	None	0.040832120925188065	0.0209805928170681	0.07946687191724777	37.32500076293945	37.14285659790039	0.5987184643745422		12.936994552612305	0.5992890000343323	4.614151477813721	true	0.11800127860501854	true	3	32	1		84.56365966796875									Y	N
93	H9	false	mtun4u	xbp1 sp	UNKNOWN	SYBR	None	0.040832120925188065	0.0209805928170681	0.07946687191724777	37.62900161743164	37.14285659790039	0.5987184643745422		12.936994552612305	0.5992890000343323	4.614151477813721	true	0.11800127860501854	true	3	32	1		84.56365966796875									Y	N
4	A4	false	3nt	xbp1 uns	UNKNOWN	SYBR	None				23.986000061035156	23.962738037109375	0.021795254200696945					true	0.257616	true	3	18	1		83.97288513183594									N	N
5	A5	false	3nt	xbp1 uns	UNKNOWN	SYBR	None				23.959999084472656	23.962738037109375	0.021795254200696945					true	0.257616	true	3	18	1		83.97288513183594									N	N
6	A6	false	3nt	xbp1 uns	UNKNOWN	SYBR	None				23.941999435424805	23.962738037109375	0.021795254200696945					true	0.257616	true	3	18	1		84.07135009765625									N	N
10	A10	false	mnt	xbp1 uns	UNKNOWN	SYBR	None				22.8799991607666	22.879777908325195	0.05090446025133133					true	0.257616	true	3	17	1		83.87442016601562									N	N
11	A11	false	mnt	xbp1 uns	UNKNOWN	SYBR	None				22.930999755859375	22.879777908325195	0.05090446025133133					true	0.257616	true	3	17	1		83.87442016601562									N	N
12	A12	false	mnt	xbp1 uns	UNKNOWN	SYBR	None				22.82900047302246	22.879777908325195	0.05090446025133133					true	0.257616	true	3	17	1		83.77595520019531									N	N
52	E4	false	3tun	xbp1 uns	UNKNOWN	SYBR	None				23.204999923706055	23.163331985473633	0.054282255470752716					true	0.257616	true	3	17	1		83.97288513183594									N	N
53	E5	false	3tun	xbp1 uns	UNKNOWN	SYBR	None				23.18199920654297	23.163331985473633	0.054282255470752716					true	0.257616	true	3	17	1		83.97288513183594									N	N
54	E6	false	3tun	xbp1 uns	UNKNOWN	SYBR	None				23.101999282836914	23.163331985473633	0.054282255470752716					true	0.257616	true	3	17	1		83.97288513183594									N	N
58	E10	false	mtun	xbp1 uns	UNKNOWN	SYBR	None				22.899999618530273	22.876028060913086	0.04782715067267418					true	0.257616	true	3	17	1		83.87442016601562									N	N
59	E11	false	mtun	xbp1 uns	UNKNOWN	SYBR	None				22.906999588012695	22.876028060913086	0.04782715067267418					true	0.257616	true	3	17	1		83.87442016601562									N	N
60	E12	false	mtun	xbp1 uns	UNKNOWN	SYBR	None				22.820999145507812	22.876028060913086	0.04782715067267418					true	0.257616	true	3	17	1		83.87442016601562									N	N
88	H4	false	3tun4uc	xbp1 uns	UNKNOWN	SYBR	None				21.81800079345703	21.81813621520996	9.115736465901136E-4					true	0.257616	true	3	16	1		83.77595520019531									N	N
89	H5	false	3tun4uc	xbp1 uns	UNKNOWN	SYBR	None				21.816999435424805	21.81813621520996	9.115736465901136E-4					true	0.257616	true	3	16	1		83.87442016601562									N	N
90	H6	false	3tun4uc	xbp1 uns	UNKNOWN	SYBR	None				21.819000244140625	21.81813621520996	9.115736465901136E-4					true	0.257616	true	3	17	1		83.87442016601562									N	N
94	H10	false	mtun4u	xbp1 uns	UNKNOWN	SYBR	None				24.18600082397461	24.20586395263672	0.026144949719309807					true	0.257616	true	3	18	1		83.67749786376953									N	N
95	H11	false	mtun4u	xbp1 uns	UNKNOWN	SYBR	None				24.195999145507812	24.20586395263672	0.026144949719309807					true	0.257616	true	3	18	1		83.57903289794922									N	N
96	H12	false	mtun4u	xbp1 uns	UNKNOWN	SYBR	None				24.236000061035156	24.20586395263672	0.026144949719309807					true	0.257616	true	3	18	1		83.57903289794922									N	N

101

102

3tungsk	actin	UNKNOWN	SYBR	None				24.485000610351562	24.45107078552246	0.0687810406088829					true	0.1807080435256173	true	3	19	1		90.18501281738281
3tungsk	actin	UNKNOWN	SYBR	None				24.496999740600586	24.45107078552246	0.0687810406088829					true	0.1807080435256173	true	3	19	1		90.08651733398438


Analysis:
For XBP1 splicing, all analyses was done with plate reader software. This is seen in the "Raw XBP1 data" tab.
Results displayed were transposed directly from these data. These results come from ddCT analysis. (unspliced XBP1 as the control)

For HSPA5 and Trib3, for replicates 1 and 2 calculations were initially done on the QPCR machine.
This provided relative quant values using the ddCT method (actin control)
As actin was run on a separate plate for the replicate 3 samples, ddCT was performed by hand. This is below:

Replicate 3 calculations:
	MEAN CT values				Relative quant (for NT)
	HSPA5	Trib3	Actin		HSPA5	Trib3	Actin
NT	20.782	28.899	24.124	NT	1	1

CA7	22.219		24.851	CA7	0.611320138846034
GSK		30.199	25.041	GSK		0.7668413298365465
NT Tun	18.503	24.702	25.817	NT Tun	15.692464063816837	59.30163596098375
								0.06792092890787867
Ca7 tun	21.429		24.863	Ca7 tun	1.0658467360679442
GSK tun		27.784	24.451	GSK tun		0.6164216886742478

For each replicate, HSPA5 and TRIB3 results were then normalized to the tunicamycin alone treatment condition results.
Pre normalization:
	1 NT	1 Tun	1 Tun CA7	1 Tun GSK	2 NT	2 Tun	2 Tun CA7	2 Tun GSK	3 NT	3 Tun	3 Tun CA7	3 Tun GSK
HSPA5	1	19.777463912963867	4.831404209136963		1	5.9979047775268555	2.0208442211151123		1	15.692464063816837	1.0658467360679442
Trib3	1	46.85881042480469		3.192003011703491	1	81.77969360351562		10.768038749694824	1	59.30163596098375		0.6164216886742478

Normalized inhibitor treatments:
	DMSO	DMSO	DMSO	CA7	CA7	CA7
HSPA5	1	1	1	0.24428835923548525	0.3369250256667757	0.06792092890787867
Trib3	1	1	1	0.06811959123089062	0.13167130219269882	0.010394682687671708
	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3


	DMSO	DMSO	DMSO	DMSO	4u8c	4u8c	4u8c	4u8c
Spliced XBP1	145.838	204.159	236.307	231.719	0.052	0.036	0.038	0.041
(Relative Unspliced)

	(ALL tunicamycin treated)
	DMSO	DMSO	DMSO	CA7	CA7	CA7	GSK	GSK	GSK
HSPA5	1	1	1	0.24428835923548525	0.3369250256667757	0.06792092890787867
Trib3	1	1	1				0.06811959123089062	0.13167130219269882	0.010394682687671708
	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3

BMDMs were derived from C57BL/6J mice and differentiated for a total of 8 days in DMEM+10% FBS + 20% L929 supernatant + sodium pyruvate + HEPES with a media addition 3 days post plating and a change of media 5 days post plating
Cells were then harvested and replated in DMEM + 10% FBS + HEPES + sodium pyruvate.
The following day, media was replaced with media containing inhibitors at 1x or received DMSO alone
2 hours post treatment, media was replaced with media containing tunicamycin and/or inhibitors (or dmso) as appropriate.
4 hours post treatment, media was removed from cells and TRIzol was added directly to wells.
RNA was then extracted from lysed samples using Direct-Zol RNA miniprep kit per manufacturers instructions (Zymo Research)
Reverse transcription was performed prior to qPCR using the iScript cDNA synthesis kit (Bio-Rad Laboratories)
Quantiative PCR was performed using Luna Universal qPCR Master Mix (New England BioLabs) using an Applied Biosystems Viia 7 Real-Time PCR system
ddCT method was used for calculating relative abundances.
The following primer sequences were used: spliced XBP1 F, 5’-ggtctgctgagtccgcagcagg-3’, R, 5’-gaaagggaggctggtaaggaac-3’ 57; unspliced XBP1 F, 5’-cagactatgtgcacctctgc-3’, R, 5’-cagggtccaacttgtccagaat-3’ 58; HSPA5 F, 5’- acttggggaccacctattcct-3’, R, 5’- atcgccaatcagacgctcc-3’ 59; Trib3 F, 5’- gcaaagcggctgatgtctg-3’, R, 5’-agagtcgtggaatgggtatctg-3’ 60–62; β-actin F, 5’- cgcagccactgtcgagtc-3’, R, 5’-ccttctgacccattcccacc-3’ 63.